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Ann Thorac Surg 2002;74:2138-2146
© 2002 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Boston MA 02115, USA
b Division of Pathology, Beth Israel Deaconess Medical Center, Boston MA 02115, USA
c Division of Biometrics Center, Beth Israel Deaconess Medical Center, Boston MA 02115, USA
d Department of Anesthesiology, Children’s Hospital, and Harvard Medical School, Boston, Massachusetts, USA
* Address reprint requests to Dr McCully, Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 77 Ave Louis Pasteur, Room 144, Boston MA 02115, USA.
e-mail: james_mccully{at}hms.harvard.edu
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: Pigs (32 to 42 kg) undergoing total cardiopulmonary bypass underwent left anterior descending coronary artery occlusion for 30 minutes. The aorta was cross-clamped and magnesium-supplemented potassium cold-blood cardioplegia (DSA; n = 6) or magnesium-supplemented potassium cardioplegia containing 50 µmol/L diazoxide (DZX; n = 6) was administered, followed by 30 minutes of global ischemia and 120 minutes of reperfusion. Left ventricular tissue samples from DSA and DZX hearts were obtained after reperfusion. Apoptosis was determined by TUNEL, caspase-3 and PARP cleavage, and caspase-3 activity. Bax and bcl-2 levels were determined and tissue morphology was examined by light and transmission electron microscopy.
RESULTS: Apoptosis, as estimated by TUNEL-positive nuclei/3,000 myocardial cells, was 120.3 ± 48.8 in DSA hearts and was significantly decreased to 21.4 ± 5.3 in DZX hearts (p < 0.05 vs control). Caspase-3 and poly-ADP-ribose polymerase cleavage and pro-apoptotic bax protein levels were significantly decreased with diazoxide (p < 0.05 vs DSA). Light and transmission electron microscopy indicated severe disruption of tissue with capillary dilatation, mitochondrial cristae damage, and evidence of increased presence of mitochondrial granules in DSA as compared with DZX hearts.
CONCLUSIONS: The addition of diazoxide (50 µmol/L) to cardioplegia significantly decreases regional myocardial apoptosis and mitochondrial damage, and provides an additional modality for achieving myocardial protection.
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Introduction |
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Previously, we have shown that magnesium-supplemented potassium cardioplegia (Deaconess Surgical Associates [DSA], Boston, MA) provides enhanced cardioprotection as compared with traditional high-potassium cardioplegia. The mechanisms by which DSA cardioplegia affords enhanced cardioprotection involve the amelioration of cytosolic, mitochondrial, and nuclear calcium overload, enhanced preservation and resynthesis of high-energy phosphates, and the modulation of nuclear and mitochondrial function [1–3]. The end effector of these mechanisms remains to be elucidated; however, recent investigations have suggested that the mitochondrial ATP-sensitive potassium (mitoKATP) channels play a predominant role in the infarct-limiting effects afforded by DSA cardioplegia [4, 5].
Previous investigations in isolated crystalloid-perfused, in situ blood-perfused animal models and in vitro studies using human trabeculae have demonstrated that nonspecific potassium channel openers such as nicorandil and pinacidil, which allow for unmodulated opening of both sarc- and mitoKATP channels, significantly enhance cardioprotection when used alone. However, these cardioprotective effects, when used in conjunction with cardioplegia, are limited or inhibited altogether [6–8].
Recently, we have investigated the cardioprotective effects of diazoxide, a cardioselective mitoKATP channel opener in conjunction with DSA cardioplegia, using a clinically relevant model of acute myocardial infarction in the in situ blood-perfused pig heart [4]. Our results indicated that the pharmacological opening of mitoKATP channels with diazoxide (50 µmol/L) with DSA cardioplegia significantly decreases infarct size (p < 0.05 vs DSA) as compared with DSA cardioplegia alone, and that these enhanced infarct-limiting effects are independent of vasodilatation [4].
At least two morphologically distinct pathways, necrosis and apoptosis, have been shown to contribute to myocardial infarct size after ischemia/reperfusion [9]. In this paper, we investigate the effects of the specific mitoKATP channel opener, diazoxide, when added to DSA cardioplegia on the modulation of apoptosis after ischemia/reperfusion, and the mechanisms modulating apoptosis in an in situ blood-perfused model of acute myocardial infarction.
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Material and methods |
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Surgical preparation
Yorkshire pigs of either gender (32 to 42 kg, n = 15) were sedated with ketamine hydrochloride (20 mg/kg, IM; Abbott Laboratories, North Chicago, IL), and anesthetized with a bolus infusion of thiopental sodium (5.0 to 7.0 mg/kg IV; Baxter Healthcare Corporation, Inc, Deerfield, IL) through an ear vein. Surgical preparation (sternotomy, tracheotomy, cannulation) was performed as previously described [4]. General anesthesia was maintained with 3.0% sevoflurane (Ultane; Abbott Laboratories, Inc) at the beginning of the surgical preparation, and then reduced to 1.0% and maintained at this concentration throughout the experiment. During cardiopulmonary bypass (CPB), propofol (0.5 to 0.7 mg/kg/min IV; Baxter Healthcare Corporation, Inc) was continuously infused through the jugular vein. Heparin sodium (5,000 IU IV; Elkins-Sinn, Inc, Cherry Hill, NJ) and 1% lidocaine (5 mL IV; Elkins-Sinn, Inc) were given before sternotomy. Heparin was administered at the same dose every 30 minutes until the end of the experiment. A silk thread (0 silk, K834H; Ethicon, Inc, Somerville, NJ) was passed around the distal third of the left anterior descending artery (LAD), and both ends of the silk tie were threaded through a small vinyl tube to form a snare [4].
Cardiopulmonary bypass
CPB was initiated at a flow rate of 75 mL/kg/min, with a two-staged cannula for venous return and the right subclavian artery for arterial inflow. To maintain hematocrit levels, CPB pumps were primed with autologous blood drawn from a donor pig [4]. All animals were allowed to stabilize for 30 minutes after cannulation for CPB and then subjected to 30 minutes of normothermic regional ischemia by tightening the snare on the distal portion of the LAD and securing with a mosquito clamp [4]. Immediately after 30 minutes of regional ischemia, the ascending aorta was cross-clamped, and cold (4°C) cardioplegia was administered antegrade through the aortic root using a cardioplegia needle (9F AR II aortic root cannula; Medtronics DLP, Inc., Grand Rapids, MI), inducing prompt diastolic arrest. DSA hearts (n = 6) received cold-blood cardioplegia (10 mL/kg). DZX hearts (n = 6) initially received DSA cardioplegia (10 mL/kg) containing diazoxide (50 µmol/L). Diazoxide was dissolved in dimethyl sulfoxide (DMSO; Fisher Scientific Co, Fair Lawn, NJ) before being added to DSA. DMSO was added as vehicle sham to DSA cardioplegia (final concentration less than 0.1%). At 15 minutes of global ischemia, an additional 5 mL/kg of DSA cardioplegia without diazoxide was readministered in both groups. All hearts received topical hypothermia during global ischemia [4]. After 30 minutes of hypothermic global ischemia, the cross-clamp and the snare from the LAD were released, and the ice packing was removed and the hearts kept on total CPB for 120 minutes of reperfusion. Control hearts (n = 3) received CPB and sham reperfusion only.
Tissue samples
Immediately after 120 minutes of reperfusion, myocardial tissue samples (approximately 15 x 30 mm each) from the area at risk (regional ischemic zone, six samples) and nonischemic area of left ventricle (global ischemic zone, six samples) consisting of epicardial, myocardial, and endocardial tissue were removed and divided into two samples. Regional ischemic and global ischemic zone samples were confirmed by monastryl blue pigment injection. The samples from each region were divided, equally and the subdivisions snap frozen, and used for either TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end-labeling), protein isolation, or histology. One subdivided sample from each zone was routinely fixed for electron microscopy with Karnovsky fixative and embedded in epoxy resin [10].
TUNEL-mediated dUTP nick end-labeling
TUNEL was performed using the ApopTag detection system (Intergen, Gaithersburg, MD) according to procedures described previously [10]. Thirty to 35 slides were selected for each sample. Five to six slides from each sample were assayed using both TUNEL and propidium iodide staining. Indirect digoxigenin fluorescence labeling of DNA was visualized as described previously [10]. Photomicrographs were taken in 10 to 15 random high-powered (20x) fields, and peroxidase TUNEL with methyl green counterstaining was performed on adjacent sequential serial slides (n = 5 to 6 for each sample) to allow for morphologic evaluation of specimens. All cells were counted on each slide, and TUNEL-positive cells were expressed per 3,000 myocardial cells [10]. Myocardial cell specificity was determined on opposite adjacent sequential serial slides (n = 5 to 6 for each sample) using the cardiac-specific monoclonal antibody for troponin I (Spectral Diagnostics Inc, Toronto, Ontario, Canada) labeled with antimouse IgG conjugated to Alexa 350 (Molecular Probes, Inc, Eugene, OR) [10]. Only those cells that could be confirmed by both peroxidase staining and Troponin I were classified as being of myocyte origin.
Western blot
Myocardial proteins were isolated on iced SDS, Nonidet NP40 lysis buffer containing protease inhibitors (2 µmol/L complete; Boehringer Mannheim; Indianapolis, IN) and cell-permeable caspase inhibitors (DEVD.fmk, 10 µmol/L, caspase-3-like irreversible inhibition; Z-VAD.fmk, 50 µmol/L, nonselective irreversible caspase inhibitor; Kamiya Biomedical Co, Seattle, WA). Protein content was determined using the Pierce BCA kit (Pierce Chemical Co, Rockford, IL). Equal amounts of protein (50 µg) were fractionated on 10% SDS-polyacrylamide gels (Invitrogen, Casbad, CA) and then electroblotted to nitrocellulose (Invitrogen). Protein equivalency and transfer efficiency were confirmed by Ponceau red staining. Membranes were blocked for 2 hours at room temperature with 5% nonfat milk in TBS (25 mmol/L Tris, 137 mmol/L NaCl, and 2.7 mmol/L KCl) containing 0.05% Tween-20, and then incubated overnight with shaking at 4°C with specific antibodies according to methods previously described [11].
Immunoblotting was performed using monoclonal antiactin clone AC-40 (1:1000 dilution; Sigma; St. Louis, MO); bax (N-20) and bcl-2 (N-19) polyclonal antibodies (1:200 dilution each; Santa Cruz Biotechnology, Santa Cruz, CA); CCP-32 (Ab-3), anticaspase-3 monoclonal antibody (1:300 dilution; Oncogene, Boston, MA); and poly-ADP-ribose polymerase monoclonal antibody (PARP, Ab-2; Oncogene, Boston, MA). After washing, the blots were incubated for 1 hour with appropriate horseradish peroxidase–conjugated antibodies, and the blots detected using ECL Plus (Amersham Pharmacia Biotech, Piscataway, NJ).
Semiquantitative analysis
Semiquantitative analysis was performed using an LKB Ultrascan XL laser densitometer (Pharmacia LKB). The integrals for each blot were calculated using the LKB GelScan XL software program for 1-D analysis [11].
Caspase-3–like activity
Protein samples for the estimation of caspase-3–like activity were isolated, separately, without caspase inhibitors. Caspase-3–like activity was estimated by colorimetric assay according to the procedures of the manufacturer (Chemicon International, Temecula, CA). Results were calibrated by standard curve using known concentrations of DEVD-pNA.
Statistical analysis
The mean ± SEM is shown for all variables. Statistical significance was determined by a mixed models analysis of variance (MM-ANOVA), which allows for both fixed and random effects and allows for more than one observation per animal (regional and global). In the analysis, when more than one observation was used from an animal, the site of ischemia (regional or global) was treated as a repeated measurement and the specific animal was incorporated as a random effect. We also tested whether different correlations for the repeated measurements in the DSA and DZX groups were necessary. All analyses were done in SAS(R) for Windows release 8.02 (SAS Institute, Cary, NC). Post hoc comparisons were made with the use of a Bonferroni correction. A p less than 0.05 was considered statistically significant.
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TUNEL
The area at risk was 13.91% ± 1.17% left ventricular (LV) mass in DSA hearts and 14.11% ± 1.79% LV mass in DZX hearts. No significant difference in area at risk was observed between groups. Apoptosis estimated by TUNEL-positive cells per 3,000 myocytes after 120 minutes of reperfusion was significantly increased (p < 0.05) in the zone of the heart made regionally ischemic by snaring of the distal portion of LAD (regional) as compared with the zone of the heart subjected only to global ischemia from aortic cross-clamping (Global) in both hearts receiving DSA cardioplegia (DSA) or DSA cardioplegia containing 50 µmol/L diazoxide (DZX). TUNEL-positive nuclei per 3,000 myocytes in the regional ischemic zones was 120.3 ± 48.8 in DSA and was significantly decreased (p < 0.05) to 21.4 ± 5.3 in DZX hearts (Fig 1).
Fewer than 20 TUNEL-positive nuclei per 3,000 myocytes were observed in global ischemic zones in DSA and DZX hearts (NS). There was no significant difference in TUNEL-positive nuclei per 3,000 myocytes between DZX regional and DSA or DZX global hearts.
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Comparison of anti- to pro-apoptotic protein (bcl-2/bax) ratios (Fig 3) indicated a significant decrease (p < 0.05) in DSA hearts as compared with control and DZX hearts in the regional ischemic zones. No significant difference from control was observed in global zones between DSA and DZX hearts as compared with control hearts; however, there was a significant increase in bcl-2/bax ratio in DZX as compared with DSA hearts .
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Comment |
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Our results indicate that the addition of the selective mitoKATP channel opener, diazoxide (50 µmol/L), to DSA cardioplegia significantly decreases apoptosis, by significantly decreasing caspase-3 cleavage and caspase-3 activity. We also show that anti-to-pro-apoptotic protein ratios are significantly decreased (p < 0.05) in DSA hearts as compared with control and DZX in the regional ischemic zones and are associated with mitochondrial structural damage.
We speculate that the decrease in apoptosis seen in our investigation is due to circulating diazoxide during reperfusion. Diazoxide is highly bound to albumin and has a plasma half-life of 22 hours [12]. These properties would allow diazoxide to provide for cardioprotection during the reperfusion phase of the regional ischemic zone. In the present report, we have used antegrade perfusion of cardioplegia and we speculate that retrograde perfusion may further limit infarct size and apoptosis.
In our investigations, 50 µmol/L diazoxide was added to DSA cardioplegia (600 mL) only during the initial administration of cardioplegia, and was not included when DSA was readministered after 15 minutes of global ischemia, based on preliminary results that showed that readministration of diazoxide resulted in a significant decrease in mean arterial pressure upon reperfusion. Diazoxide (50 µmol) in the initial administration of cardioplegia followed by cardioplegia without diazoxide in all subsequent readministrations had no effect on heart rate, mean arterial pressure, or coronary flow as compared with DSA [4]. The circulating concentration of diazoxide was not measured in this study but based upon blood volumes of 3 to 4 L in the pig. A circulating diazoxide concentration of approximately 7.5 to 10 µmol was present. This concentration of diazoxide and the effects seen in our study are in agreement with the investigations of Garlid and associates [13, 14], who have shown that diazoxide decreases cell injury in a dose-dependent manner at concentration between 1 and 30 µmol/L.
Garlid and associates have suggested that the mechanism by which diazoxide modulates infarct size involves the regulation of mitochondrial volume and electron transport, and that these mechanisms are preeminent in maintaining mitochondrial function in the intact myocardium, allowing for enhanced preservation of adenine nucleotides during ischemia and efficient energy transfer during reperfusion [13–15]. It has also been suggested that mitochondrial membrane depolarization caused by K+ entry through the opening of mitoKATP channels reduces mitochondrial Ca2+ entry through the calcium uniporter, and in turn acts to reduce mitochondrial Ca2+ overload [16]. Subsequently, these events are believed to result in ATP production and cell salvage [17].
Support for these putative mechanisms comes from studies indicating that increased mitochondrial Ca2+ accumulation destabilizes the inner mitochondrial membrane, causing the inner membrane pores to open, which permits further movement of cations across the mitochondrial membrane [17]. The opening of these pores renders the mitochondrion incapable of synthesizing ATP, suggested to be a key event in the process leading to myocardial cell death. This would agree with our earlier reports, in which we suggested that modulation of mitochondrion "futile calcium cycling" may be of significance in the modulation of ischemic myocardial injury [3]. This hypothesis has been supported by Pacher and Hajnoczky [18], who have shown that mitochondrial Ca2+ uptake is crucial for the initiation of apoptosis but can be modulated by antiapoptotic proteins (bcl-2).
The expression of bcl-2–related proteins has been shown to be a key element in the genesis of apoptosis and is known to affect mitochondrial function and regulate the release of apoptosis-activating factors [19]. Recent investigations have shown that ATP depletion in cells initiates the translocation of bax, a pro-apoptotic bcl-2 family member protein, from the cytosol to the outer mitochondrial membrane, by mechanisms that remain unclear [20]. The translocation of bax is thought to cause mitochondrial dysfunction and swelling, and to induce the efflux of cytochrome c to the cytosol by a mechanism that is thought to involve changes in mitochondrial permeability caused by the opening of the permeability transition pore in the mitochondrial inner membrane, or mitochondrial hyperpolarization followed by swelling and membrane rupture [21]. Cytochrome c, on release to the cytosol, activates caspases, normally expressed as latent zymogens, which are cleaved and reassociated to generate catalytically active heterodimers.
Bcl-2 is an antiapoptotic protein that inhibits the oligomerization of bax and prevents apoptosis by preserving mitochondrial integrity and suppressing cytochrome c release without ameliorating ATP depletion [22]. It is the activation of the effector caspase, caspase-3, responsible for the cleavage of poly ADP-ribose polymerase (PARP), a nuclear enzyme that is catalytically activated by DNA strand interruptions, that is the common event initiated by the multiple different stimuli that induce apoptosis [22].
Our results show that anti-to-pro-apoptotic protein (bcl-2/bax) ratios are significantly decreased (p < 0.05) in DSA hearts as compared with control and DZX hearts in the regional ischemic zones, and that there was a significant increase in the anti-to-pro-apoptotic protein ratio in DZX as compared with DSA hearts in the global ischemic zone. We further show that diazoxide preserved mitochondrial structure. These results are in agreement with the proposed mechanism of apoptotic signal transduction and mitochondrial structure in the apoptotic cascade [19–22].
Recently, Ozcan and associates [23] have suggested that treatment of mitochondria with diazoxide during the anoxic period effectively preserves the structural and functional integrity of mitochondria, maintaining ATP generation after the anoxic insult. This would agree with our observations using transmission electron microscopy, which show extensive swelling of the mitochondrial matrix and disruption of cristae in the regional ischemic zone of DSA hearts, which is less pronounced in the global ischemic zone in DSA hearts. Only a slight swelling of the intercristae matrix was observed in either the regional or global ischemic zones in DZX hearts.
We also observed large electron-opaque mitochondrial granules in DSA hearts. These granules are also present in DZX hearts, but are significantly smaller in size and significantly less numerous than those observed in DSA hearts. Previous investigations have shown that these granules contain calcium and inorganic phosphate [24]. When modest amounts of calcium ions are present, they are deposited in these granules; however, if a large increase in calcium occurs de novo, granule formation occurs [24]. The increased size and number of granules seen in DSA hearts as compared with DZX hearts would suggest that there is an increased accumulation of mitochondrial Ca2+ in DSA hearts that is attenuated in DZX hearts. This would agree with the recent observations of Murata and associates [25], who have shown that opening mitoKATP and partial mitochondrial membrane depolarization attenuates mitochondrial Ca2+ overload.
Whereas the mechanism(s) by which diazoxide effects infarct limitation remains to be elucidated, our results indicate that at least in part, the addition of diazoxide to DSA cardioplegia significantly decreases apoptosis through the modulation of bcl-2/bax ratio and by decreasing caspase-3 activity and preserving mitochondrial structure in the in situ blood-perfused pig heart model of acute myocardial infarction, and represents an additional modality for enhancing myocardial protection.
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Discussion |
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DR McCULLY: We measured coronary flow in this study using both proximal and distal probes. There was no difference in mean arterial pressure or in coronary flow between groups in either proximal, distal, or proximal to distal coronary flow. We used a very low level of diazoxide, 50 µmol, in a 4-to-1 solution of cardioplegia, of approximately 500 mL. If we calculate the blood volume in a pig to be about 4 L, the final concentration was 6 to 8 µmol. This concentration is well below the concentration needed to induce vasodilation.
It should be noted that we included diazoxide only in the first delivery of cardioplegia. We found that if diazoxide was included also in the second delivery of cardioplegia or during reperfusion, there were significant increases in mean arterial pressure. However, if 50 µmol diazoxide is given once only time, these events do not occur.
DR RALPH I. DAMIANO (St Louis, MO): Our lab has had a long interest in potassium channel openers, though we have not focused, as your group has, on the mitochondrial potassium channel openers. We have noticed with the nonspecific KATP channel openers a significant toxicity at higher doses. My first question is whether you noticed any dose-response effect of diazoxide, and is there toxicity like you see with the nonspecific openers?
Also, you inferred that there was lower intracellular calcium or mitochondrial calcium levels. I wondered if you have had the opportunity to actually measure intracellular mitochondrial calcium with this agent and whether you saw a difference?
And finally, do the beautiful results you have shown with apoptosis and histology translate into significant functional benefits either regionally or globally? I know you have shown fantastic functional recovery in your prior studies with magnesium-enhanced cardioplegia, so does this make it even better?
DR McCULLY: Yes, we have done concentration studies based on the studies of Dr Keith Garlid. We have have used 50 and 100 µmol concentrations, and we have found that at 100 µmol, we see changes in mean arterial pressure and reentry ventricular fibrillation. When we used nicorandil, as you have used earlier on, we consistently observed ventricular fibrillation on reperfusion. The use of 50 µmol diazoxide resulted in no change in mean arterial pressure and no reentry ventricular fibrillation.
As far as measuring the calcium level, we have measured cytosolic, mitochondrial, and nuclear calcium previously with DSA cardioplegia, and we have shown that there was an increase in all these levels during ischemia that was partially alleviated with the addition of magnesium, resulting in decreased high-energy phosphate depletion through futile calcium cycling.
In this study, we have not directly measured mitochondrial calcium, but our studies using transmission electron microscopy show numerous large electron-dense granules. It has been shown by others that these granules contain calcium and that with a moderate increase in mitochondrial calcium, the calcium is stored in the granules present in the mitochondria and they become electron dense. If mitochondrial calcium is significantly increased, the number of granules increases to allow for greater calcium storage and the granules are increased in size. This is exactly what we see in our experiments, and thus we speculate that we have increased mitochondrial calcium accumulation.
With regard to function, in previous studies, we have shown that myocardial infarct is modulated by the mitochondrial KATP channels, whereas function is modulated by the sarcolemmal KATP channels. DSA cardioplegia already provides almost optimal recovery, and we achieve approximately 95% functional recovery at 2 hours of reperfusion after 30 minutes of regional ischemia and 30 minutes of global ischemia.
In this study, we only went to 2 hours of reperfusion, and our last functional measurements (PRSW, Tau, SS, Sb, PSSO) were obtained at 90 minutes of reperfusion. We found that only postsystolic shortening was increased when diazoxide was added to DSA cardioplegia, but this did not reach statistical significance. We believe that if we extended the reperfusion time, we would find significant increases primarily due to the benefits from tissue preservation.
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