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Ann Thorac Surg 2004;77:1384-1389
© 2004 The Society of Thoracic Surgeons

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Original article: cardiovascular

Improved profile of bad phosphorylation and caspase 3 activation after blood versus crystalloid cardioplegia

^a Division of Cardiothoracic Surgery, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

Accepted for publication September 10, 2003.

^* Address reprint requests to Dr Sellke, Division of Cardiothoracic Surgery, BIDMC, LMOB 2A, 110 Francis St, Boston, MA 02215, USA.
e-mail: fsellke{at}caregroup.harvard.edu

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
BACKGROUND: Expression of Bcl-2 family proteins and activation of terminal caspase 3 are important for ischemia-reperfusion-induced apoptosis. Bad and Bax are pro-apoptotic proteins, whereas, phosphorylation of Bad inhibits its binding to and inactivation of anti-apoptotic Bcl-2. Thus, decreases in phospho-Bad would be proapoptotic. We investigated if blood (BCP) or crystalloid cardioplegia (CCP) differentially affects apoptosis gene-related proteins.

METHODS: Rabbit hearts were perfused with Krebs-Henseleit buffer (KHB) on a Langendorff apparatus. Control hearts (n = 6) were perfused for 90 minutes without cardioplegic ischemia. In the other two groups, hearts were arrested for 30 minutes (37°C) with BCP (n = 6) or with CCP (n = 6) administered continuously (1.5 mL/min). The hearts were reperfused for 30 minutes with KHB. Left ventricle (LV) performance was measured before cardioplegic arrest and at 30 minutes of reperfusion. In vitro relaxation responses of precontracted microvessels (100–180 µm) were obtained in a pressurized no-flow state. Total and activated or phosphorylated caspase 3, Bcl-2, Bad, and Bax were measured by quantitative immunoblotting using specific antibodies.

RESULTS: Blood cardioplegia significantly improved the recovery of LV developed pressure compared to CCP (p < 0.05). The endothelium-dependent relaxation in response to adenosine 5'-diphosphate was greater after BCP than after CCP (59.9 ± 4% vs 26.9 ± 6%, respectively; p < 0.05). There were no differences in total protein levels of caspase 3, Bcl-2, Bad, and Bax between the groups. Both BCP and CCP increased caspase 3 activity as compared with controls, but CCP caused more activation of caspase 3 than BCP (6.2 ± 0.7 fold vs 3.1 ± 0.4, p < 0.05). Both BCP and CCP induced phosphorylation of Bad at Ser¹¹², but BCP caused greater phosphorylation of Bad (3.5 ± 0.2 fold vs 2.0 ± 0.12 fold, respectively, p < 0.05) than CCP.

CONCLUSIONS: Blood cardioplegia is superior to CCP in inhibiting the activation of caspase 3 and in increasing phospho-Bad. These actions of BCP were associated with improved LV function and endothelium-dependent relaxation of coronary microvessels. These results may provide molecular mechanisms by which to improve myocardial protection during cardiac surgery.

	Introduction

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Crystalloid cardioplegia has been routinely used for several decades to achieve cardiac arrest and to protect the myocardium during open-heart surgery. However, myocardial and vasomotor dysfunction still occur after hyperkalemic crystalloid cardioplegia [1, 2]. The addition of blood to a crystalloid cardioplegic solution can improve the recoveries of vasomotor and myocardial function after hyperkalemic cardioplegic arrest [2–4]. Although blood cardioplegia has decreased the mortality and morbidity associated with cardiac surgery, the underlying mechanism of decreasing ventricular and vasomotor dysfunction is not fully understood. Recent studies suggest that apoptosis may be an important mechanism in postoperative myocardial dysfunction [5, 6].

Apoptosis is a genetically controlled, programmed cell death and results in single cell loss with characteristic cell shrinkage, membrane—blebing, DNA-fragmentation, and chromatin-condensation but no increase in plasma membrane permeability [7]. Apoptosis of endothelial and cardiac cells was observed in ischemia-reperfusion injury [8–10]. However, the mechanisms underlying ischemia-reperfusion-induced apoptosis are poorly understood. Several distinct signaling pathways have been proposed to be responsible for apoptosis. One main pathway of apoptosis in myocytes is by ischemia-reperfusion-induced expression of Bcl-2 family of proteins and activation of pro-apoptotic terminal caspase 3 [11]. Bad and Bax are pro-apoptotic Bcl-2-family-protein, whereas, phosphorylation of Bad inhibits its binding to, and inactivation of, anti-apoptotic Bcl-2. Thus, increases in phospho-Bad and(or) expression of Bcl-2 would be antiapoptotic [12].

Based on this observation, we hypothesized that blood cardioplegia may be superior to crystalloid cardioplegia in inhibiting the expression of pro-apoptotic Bcl-2-family-protein and the activation of cysteine protease family of caspases. We investigated whether blood or crystalloid cardioplegia differentially affects apoptosis gene-related proteins, left ventricular (LV), and microvascular endothelium function in the isolated, and perfused rabbit heart exposed to ischemia-reperfusion injury.

	Material and methods

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Experimental model
Animals were cared for in accordance 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" (National Institutes of Health publication No. 5377–3, 1996). The Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center and Harvard Medical Area Standing Committee approved the protocols used in this study.

New Zealand white rabbits (1.5 to 2.5 kg) were used in this study. Rabbits were anesthetized with ketamine (35 mg/kg) and xylazine (2.5 mg/kg, intramuscular [IM]), anticoagulant with heparin (2000 U/kg, intravenous [IV]) and the heart was rapidly exposed. The aorta was cannulated and the heart was retrogradely perfused in situ to avoid ischemia. The heart was then excised and mounted in an organ chamber on a Langendorff perfusion system. The heart was retrogradely perfused at 75 mm Hg with a modified Krebs-Henseleit buffer (KHB) with the following composition (mmol/L): NaCl 118, NaHCO₃ 25, KHPO₄ 1.2, KCl 4.7, MgSO₄ 1.2, CaCl₂ 1.8, and glucose 11.0. The Krebs-Henseleit buffer was equilibrated with 95% O₂ and 5% CO₂, adjusted to a pH of 7.35 to 7.4 at 37°C and filtered with a 5 µm filter (Gilman Scientific, Inc, Ann Arbor, MI). Right ventricular myocardial temperature was measured with a thermistor needle probe (Mallinckrodt, Inc, St. Louis, MO) and was maintained at 37°C throughout the experiment by regulation of the organ chamber temperature. Our Langendorff apparatus permits instantaneous change of the perfusion fluids from standard KHB to one containing different pharmacological substances or cardioplegia solution by adjusting an inlet valve to the aortic perfusion cannula.

Measurements
Mean coronary flow (CF mL/min) was measured by timed collection of effluent from the right ventricle exiting the heart from the severed pulmonary artery. Isovolumetric measurement of LV performance was made using a compliant latex balloon connected to a pressure transducer, inserted in the LV across the mitral valve. A calibrated syringe attached to the pressure transducer system was used to fill the balloon with a volume of saline needed to maintain a LV end-diastolic pressure (LVEDP) of 5 mm Hg during measurement of baseline LV performance. This same balloon volume was used for subsequent measurements of left ventricular performance after reperfusion. Left ventricular performance was assessed by measurement of LV developed pressure (LVDP, mm Hg) and left ventricular end-diastolic pressure (LVEDP, mm Hg). Positive and negative first derivatives of LVDP (+dP/dt and −dP/dt, mm Hg/s) were calculated as indices of ventricular contractility and compliance, respectively. Analog pressure data from the LV balloon were amplified and converted to a digital signal for on-line data recording and computation (Gould-PONEMAN, Gould, Valley View, OH). Continuous pressure measurements were sampled at specific time points in each experiment. Hearts failing to generate a LVDP more than 80 mm Hg, or a CF less than 25 mL/min during the stabilization phase of the experiment were excluded from further study.

Experimental protocols
After 30 minutes of equilibration, hearts were then divided into three groups. The control group was further buffer-perfused for 60 minutes without cardioplegic ischemia. In the other two groups, hearts were randomized to receive cardioplegic infusion by either crystalloid or blood before the onset of 30 minutes of normothermic ischemia. In hearts receiving crystalloid cardioplegia, a 50 mL bolus of warm crystalloid cardioplegia solution was administered and then followed by a continuous infusion of 1.5 mL/min over the ischemic period. In the blood cardioplegia group, hearts were initially infused with 50 mL of warm blood cardioplegia solution and then followed by a continuous infusion of 1.5 mL/min over the period of ischemic arrest. The composition of the crystalloid cardioplegic solution was (in mmol/L): NaCl, 121; KCl, 25; NaHCO₃, 12; and glucose, 11.1 (pH = 7.6 and partial pressure of oxygen range = 180 to 300 mm Hg). Blood cardioplegic solution consisted of equal volumes of crystalloid cardioplegic solution and blood removed from the rabbit. Potassium chloride was supplemented to raise the K⁺ concentration to 25 mmol/L.

After a total of 30 minutes of cardioplegia arrest, the aortic cross clamp was removed and the heart was reperfused for 30 minutes with KHB. The hearts were excised and cut into two pieces. One piece of LV tissue was immersed in cold KHB buffer for in vitro microvessel study. The other was put into liquid nitrogen and stored at −80°C for the protein analysis. Myocardial samples were also taken from KHB-perfused control hearts for molecular comparisons.

In vitro coronary microvessel studies
Coronary artery microvessels (100 to 180 µm in internal diameter) from the LV were dissected by using a 10x to 60x microscope (Olympus Optical, Tokyo, Japan). Microvessels were placed in a microvessel chamber, cannulated with dual glass micropipettes measuring 40 to 80 µm in diameter, and secured with 10 to 0 nylon monofilament sutures (Ethicon, Inc, Somerville, NJ). Oxygenated (95% oxygen and 5% carbon dioxide) Krebs buffer solution warmed to 37°C was continuously circulated through the microvessel chamber. The vessels were pressurized to 40 mm Hg in a no-flow state by using a burette manometer filled with a Krebs buffer solution. With an inverted microscope (40x to 200x Olympus CK2, Olympus Optical) connected to a video camera, the vessel image was projected onto a black and white television monitor. An electronic dimension analyzer (Living System Instrumentation, Burlington, VT) was used to measure the internal lumen diameter. Vessels were allowed to bathe in the organ chamber for at least 30 minutes before a pharmacologic intervention.

Immunoblotting
Total lysate from tissue was obtained as previously described [13]. Total protein was fractionated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation, Bedford, MA) using a semidry transfer apparatus (Millipore). Membranes were stained with Ponseau S, and then incubated with 5% nonfat dry milk in 50 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, and 0.1% Tween 20 (TBST) buffer for 1 hour at room temperature, to block nonspecific binding. Membranes were incubated with rabbit polyclonal anti-Bcl-2, Bad, and Bax (Cell Signaling Tech. Inc, Beverly, MA) 1:1000 (v/v) dilution in 2.5% nonfat dry milk, mouse monoclonal antiphospho-Bad (Cell Signaling Tech. Inc, Beverly, MA) 1:2000 (v/v) dilution with 2.5% bovine serum albumin, rabbit polyclonal anticaspase 3 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) 1/1000 (v/v) for 1 hour at room temperature. After being washed with TBST, the membranes were incubated for 1 hour in 2.5% nonfat dry milk in TBST containing 1:3000 diluted with the appropriated secondary antibody of either a sheep anti-rabbit or sheep anti-mouse immunoglobulin G (Jackson Immunolabs, West Grove, PA), at 1:3000 (v/v) dilution conjugated to horseradish peroxidase. Peroxidase activity was visualized by means of an enhanced chemiluminescence (ECL) substrate system and exposed to roentgenogram films (Amersham, Arlington Heights, IL). The intensities of bands were determined by the National Institute of Health image program (NIH Image 1.6).

Drugs
U46619, sodium nitroprusside, and adenosine 5-diphosphate were obtained from Sigma Chemical (St. Louis, MO). All drugs were dissolved in ultrapure distilled water. All solutions were prepared on the day of the study.

Data analysis
Data were presented as the mean and standard error of the mean. The relaxation responses were expressed as the percentage of relaxation of the U46619-preconstricted diameter of the microvessels. The dose-response curves of all experimental groups were compared using two-way analysis of variance with a repeated-measures design (followed by the Student-Neumann-Keuls test, SigmaStat, SPSS Inc, Chicago, IL). Statistical significance was taken at a p value of less than 0.05. The paired Student's t test was used to compare protein expressions and changes in homodynamic variables after reperfusion between groups.

	Results

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LV function and coronary flow
There were no significant differences in baseline LV performance before ischemia between blood and crystalloid cardioplegia. The recoveries of LVDP (p < 0.05), +dP/dtmax (p < 0.05) and −dP/dtmax (p < 0.05) were significantly greater in blood cardioplegia (BCP) perfused hearts than in crystalloid cardioplegia (CCP) perfused hearts (Table 1). There were no significant differences in recovery of CF between the BCP and CCP.

View larger version (11K): [in this window] [in a new window]   Fig 1. Response to SNP in vitro of rabbit coronary microvessels from control hearts (n = 6, •) and hearts after 30 minutes of ischemic arrest using CCP (n = 6, ) or BCP (n = 6, ) followed by 30 minutes of reperfusion. (BCP = blood cardioplegia; CCP = crystalloid cardioplegia; SNP = sodium nitroprusside.)

View larger version (15K): [in this window] [in a new window]   Fig 2. Response to ADP in vitro of rabbit coronary microvessels from control hearts (n = 6, •) and hearts after 30 minutes of ischemic arrest using CCP (n = 6, ) or BCP (n = 6, ) followed by 30 minutes of reperfusion. *p < 0.05 BCP versus CCP. (ADP = adenosine 5'-diphosphate; BCP = blood cardioplegia; CCP = crystalloid cardioplegia.)

View larger version (28K): [in this window] [in a new window]   Fig 3. Caspase 3 cleavage: protein was obtained from control hearts and hearts after 30 minutes of ischemic arrest using CCP or BCP followed by 30 minutes of reperfusion. (A) Representative blots showing proform (32 kda) and p17 subunit of caspase 3 protein levels in different groups. (B) Graph showing the fold-increase of p17 subunit of caspase 3 protein levels in different groups; n = 6 for each group *p < 0.05, **p < 0.01 compared with control; p < 0.05 compared with CCP. (BCP = blood cardioplegia; CCP = crystalloid cardioplegia.)

View larger version (25K): [in this window] [in a new window]   Fig 4. Western blots showing total Bcl-2 (A) and Bax (B) protein levels from control hearts and hearts after 30 minutes of ischemic arrest using CCP or BCP, followed by 30 minutes of reperfusion. Graph showing the fold-increase in Bcl-2 (A) and Bax (B) protein expression in different groups; n = 6 for each group. (BCP = blood cardioplegia; CCP = crystalloid cardioplegia.)

View larger version (24K): [in this window] [in a new window]   Fig 5. Phosphorylation of Ser112 of the pro-apoptotic protein Bad from control hearts and hearts after 30 minutes of ischemic arrest using CCP or BCP followed by 30 minutes of reperfusion. (A) Western blots showing total and phosphorylated Bad protein levels. (B) Graph showing the fold-increase in Bad phosphorylation in different groups; n = 6 for each group, *p < 0.05, **p < 0.01 compared with control, p < 0.05 compared with CCP. (BCP = blood cardioplegia; CCP = crystalloid cardioplegia.)

	Comment

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Our previous studies, and those of others, have found that microvascular endothelium and LV function were impaired after exposure to crystalloid cardioplegia in pigs [2–4]. The present data confirmed those previous findings, indicating hyperkalemic crystalloid cardioplegia severely reduced the recoveries of LV function and microvascular endothelium-dependent relaxation after ischemic arrest and reperfusion. The poor oxygen-carrying capacity, low osmotic property and buffers, low endogenous nutrients, and antioxidant capacity of crystalloid may contribute to its detrimental effects following ischemia and reperfusion injury [14]. The present data are also consistent with those previous findings, indicating that addition of blood to crystalloid cardioplegia preserved cardiac function [2–4, 15]. Blood-based cardioplegia, although not completed preserving endothelium-dependent relaxation, is more effective than a purely crystalloid-based solution in the isolated heart preparation.

Recent study indicates that apoptosis is induced in the human cardioplegic-reperfused heart, suggesting apoptosis may play an important role in myocardial dysfunction after open-heart surgery [5, 6]. More recently, studies by Yeh and colleagues [16] demonstrated that cold crystalloid cardioplegia infusion in pigs during CPB impaired the morphologic integrity of the coronary endothelium and induced cardiomyocyte apoptosis. Furthermore, the present studies examined the effect of cardioplegia on apoptosis gene-related proteins in rabbits.

The Bcl-2 family of proteins regulates apoptosis by controlling mitochondrial permeability and the release of cytochrome C [6, 17, 18]. The anti-apoptotic proteins Bcl-2 reside in the outer mitochondrial wall and inhibit cytochrome C release. The pro-apoptotic Bcl-2 proteins, such as Bad and Bax, reside in the cytosol, but translocate to mitochondria and form a pro-apoptotic complex with Bcl-2. This translocation is inhibited by survival factors that induce phosphorylation of Bad, leading to its cytosolic sequestration. Thus, phosphorylation of Bad may promote cell survival [12]. Steenbergen and colleagues [12] recently found that a significant decrease in phospho-Bad (Ser¹¹²) in failing hearts and this action was also consistent with a pro-apoptotic shift in heart failure. Baines and colleagues [19] reported that activation of protein kinase C-/extracellular regulated proteins was associated with phosphorylation of Bad at Ser¹¹². Bad can be phosphorylated on Ser¹¹², Ser¹³⁶, Ser¹⁵⁵, and(or) Ser¹⁷⁰, and the relative importance of phosphorylation at different sites is still unclear [12].

The ratio of Bcl-2/Bax protein has also been suggested to determine survival or death after ischemia and reperfusion [20–23]. Zhao and colleagues [20, 21] reported that ischemia and reperfusion significantly decreased the expression of Bcl-2 and increased the expression of Bax in the ischemic-reperfused myocardium. In contrast, we did not find any change in the expression of Bcl-2, Bad, and Bax proteins in the cardioplegic-reperfused heart. Instead, we found that Bad was phosphorylated at Ser¹¹² following blood and crystalloid CP, but blood caused greater phosphorylation of Bad at Ser¹¹² than crystalloid CP. This shows that cardioplegic arrest and reperfusion may alter the phosphorylation of the Bcl-2 family of proteins. They suggest that changes in phosphorylation or translocation of the Bcl-2 family of proteins, rather than in its total proteins, may be the primary indicators that apoptosis is induced. The enhanced phospho-Bad by blood cardioplegia may contribute to its anti-apoptotic actions.

We also observed that terminal caspases are activated after cardioplegic arrest and reperfusion. The active p17-subunit of caspase 3 was detected after 30 minutes of reperfusion, indicating proteolytical activation of caspase 3. Crystalloid CP caused more activation of caspase 3 than blood CP. This is in agreement with recent reports, which demonstrate that causal involvement of caspase 3 in ischemia-reperfusion induced apoptosis [24, 25].

In conclusion, BCP is superior to CCP in inhibiting the activation of caspase 3 and in increasing phospho-Bad. These actions of BCP were associated with an improved LV function and endothelium-dependent relaxation of coronary microvessels. These results may provide molecular mechanisms by which BCP improves myocardial protection during cardiac surgery.

	Acknowledgments

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This research project was supported in part by the National Heart, Lung and Blood Institute, HL-69024 and HL-46716. We thank Dr Tanveer Khan for his help in preparing the abstract of this manuscript.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Frank W. Sellke Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Feng, J. Articles by Sellke, F. W. Search for Related Content PubMed PubMed Citation Articles by Feng, J. Articles by Sellke, F. W. Related Collections Myocardial protection