Postconditioning has been verified to provide cardioprotection and is associated with the state of mitochondrial permeability transition pore. However, there are a few limitations with clinical use of classic postconditioning; therefore, the purpose of this investigation was to study whether inhibition of mitochondrial permeability transition pore opening with cyclosporine A also provided cardioprotection. Langendorff-perfused Sprague–Dawley rat hearts were perfused for 20 minutes with Krebs–Henseleit buffer followed by 30 minutes of crystalloid cardioplegia and 60 minutes of reperfusion. Control hearts (Con group) were reperfused with Krebs– Henseleit buffer. Postconditioning hearts (Ipo group) were with six cycles of 10 seconds reocclusion separated by 10 seconds perfusion before reperfusion. Cyclosporine A postconditioning hearts (CsA group) were reperfused with Krebs–Henseleit buffer containing 0.8 μmol/L cyclosporine A at first 5 minutes of reperfusion. Compared with Con group, myocardial performance was better preserved in CsA group. Mitochondrial outer membrane integrity was preserved, with less cytosolic diffusion of cytochrome C (p < 0.05) and less frequency of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate (dUTP) nick end labeling–positive myocytes in Ipo and CsA group (p < 0.05). Postconditioning prevented apoptosis-related mitochondrial permeabilization and dysfunction after cardioplegic arrest. Cyclosporine A postconditioning had a better effect than classic postconditioning in myocardial performance.
From the †Fuwai Hospital and Cardiovascular Institute, and ‡Department of Cardiopulmonary Bypass, Fuwai Hospital and Cardiovascular Institute, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, People's Republic of China.
Submitted for consideration July 2010; accepted for publication in revised form November 2010.
Reprint Requests: Cun Long, MD, Fuwai Hospital and Cardiovascular Institute, Chinese Academy of Medical Sciences, Peking Union Medical College, 167 Bei li shi Road, Fuwai Da Jie, Xi Cheng District, Beijing 100037, People's Republic of China. Email: firstname.lastname@example.org.
Early reperfusion is the definitive treatment to reduce the infracted area, the left ventricular contractile dysfunction, and apoptosis. However, reperfusion itself carries the potential risk of worsening the tissue damage after ischemia—a phenomenon known as reperfusion injury.1 Accumulating evidence suggests that reperfusion may cause irreversible myocardial injury, possibly through a form of mitochondrial dysfunction that had been designated permeability transition.2–4 By opening in the first few minutes of reperfusion, the mitochondrial permeability transition pore (mPTP) mediated reperfusion- induced myocyte death.5 In 2003, Zhao et al.6 found a phenomenon named ischemic postconditioning that was described as brief and repeated episodes of ischemia reperfusion immediately after a period of sustained ischemia, producing less myocardial injury. Some studies7,8 confirmed that ischemia postconditioning was related to inhibition of mPTP opening. The mPTP is a large conductance pore of the inner mitochondrial membrane, which on opening renders the otherwise impermeable inner membrane, freely permeable to solutes up to 1,500 Da in size.9 The opening of mPTP results in swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane, leading to the translocation of cytochrome C and other proapoptotic factors into the cytosol, which initiates the apoptotic death pathway.10 Cyclosporine A, which acts by inhibiting the peptidyl-prolyl cis-trans isomerase activity of cyclophilin D, is a potent inhibitor of mitochondrial permeability transition, and several reports indicated that it could limit ischemia reperfusion injury under experimental conditions.2,11,12 The purpose of this study was thus to confirm whether classic postconditioning and cyclosporine A postconditioning can protect myocardium that suffered 30 minutes of global ischemia followed by 60-minute reperfusion, through the aspects of cardiac function, translocation of cytochrome C, measurement of mPTP opening, and myocyte apoptosis.
Materials and Methods
The experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health (NIH Publication No. 85-23, revised 1996) and approved by the Research Commission on Ethics of Fu wai hospital.
Male Sprague–Dawley rats (weighing 350–450 g, n = 36) were used and anesthetized with pentobarbital (50 mg/kg), intraperitoneally. Heparin (1,000 UI/kg) was injected into the femoral vein. Hearts were rapidly removed and placed in ice-cold buffer, aorta was canulated, and hearts were perfused in Langendorff-perfusion apparatus mode using Krebs-Henseleit bicarbonate buffer (K-H, containing in mmol/L: glucose 11.0, NaCl 118.5, KCl 4.75, MgSO4 1.19, KH2PO4 1.18, NaHCO3 25.0, and CaCl2 1.4) at pH 7.4. The buffer was bubbled with 95% O2/5% CO2 at 37°C, and perfusion was performed under a hydrostatic pressure of 100 cm H2O.
Three groups of hearts were perfused for a 20-minute stabilization period followed by 30 minutes of crystalloid cardioplegic arrest and 60 minutes of reperfusion. During the stabilization period, all the groups were perfused with K-H buffer. Control hearts (Con group, n = 8) were perfused with K-H buffer alone at reperfusion period. Cyclosporine A postconditioning hearts (CsA group, n = 8) were perfused with K-H buffer containing cyclosporine A (0.8 μmol/L)13 for the first 5 minutes of reperfusion, then perfused with K-H buffer. Postconditioning hearts (Ipo group, n = 8) before reperfusion were with six cycles of 10 seconds reocclusion separated by 10-second reperfusion. Cardioplegic arrest for the three groups was induced with crystalloid cardioplegia (60 ml/kg, St. Thomas II cardioplegia containing in mmol/L: NaCl 100.0, NaHCO3 10.0, KCl 16.0, MgCl2·6H2O 16.0, CaCl2·2H2O 1.2, pH 7.40 ± 0.5). Cyclosporine A was purchased from Genmed Scientifics Inc. (Genmed Scientifics Inc., MA)
Another 12 rats were prepared to test the rate of swelling of mitochondria (four rats for each group). At the baseline and 5 minutes of reperfusion, left ventricular tissues were harvested for the test.
Cardiac Function Measurements
Isovolumetric measurement of left ventricular performance was made with a water-filled balloon connected to a pressure transducer (AD Instruments, Colorado Springs, CO) and inserted into the left ventricle across the mitral valve. The volume of the water-filled balloon was adjusted to an end- diastolic pressure below 10 mm Hg and kept constant throughout the entire experiment. Left ventricular performance was assessed with heart rate, left ventricular developed pressure (LVDP), and maximum positive and negative derivative of left ventricular pressure (±dP/dtmax). Analog data (heart rate, LVDP, and ±dP/dtmax) were digitalized and analyzed (Chart IV; AD Instruments, Colorado Springs, CO). Hemodynamic data were compared at comparable time points in all three groups.
Isolation of mitochondrial and cytosolic proteins was performed using the Mitochondria/cytosol Fractionation Kit (Beyotime Inst. Biotech, Peking, China). Myocardium samples were mechanically homogenized and lysed. Unlysed cells and nuclei were removed by centrifugation at 600g (5 minutes × 2 at 4°C). The supernatant was spun at 11,000g (10 minutes at 4°C), and a small sample of the supernatant (cytosol) and the pellet were kept for Western blot and measurement of mPTP opening, respectively.
Cytochrome C Release
Mitochondrial membrane permeability was evaluated by measurement of cytochrome C released into the cytosol. After subcellular fractionation, cardiac cytosolic samples were standardized for protein concentration by bicinchoninic acid assay, denatured, and subsequently transferred to nitrocellulose membranes. The membrane was blocked with 5% nonfat milk and incubated overnight at 4°C with specific anti-rabbit cytochrome C (1:500; Cell Signaling Technology, CA). For protein loading control, membranes were stripped and reprobed with anti-mouse Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (1:1,000; Santa Cruz Biotechnology, Inc., CA). Secondary antibodies were coupled to horseradish peroxidase (anti-rabbit IgG 1:1,000; anti-mouse IgG 1:2,000 Santa Cruz Biotechnology, Inc., CA). Cytosolic cytochrome C was normalized to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH).
Measurement of mPTP Opening
The opening of the mPTP was determined at 25°C under deenergized conditions by following the decrease in light scattering monitored as A520 (Beckman coulter DU730, CA) that accompanies mitochondrial swelling. The mitochondrial protein concentration used, determined using the Bradford assay, was 0.2 mg/ml. The buffer for deenergized conditions was KSCN 150 mM, Mops 20 mM, Tris 10 mM, nitrilotriacetic acid 2 mM, A23187 2 μM, rotenone 0.5 μM, antimycin A 0.5 μM, pH 7.2, and CaCl2 added at 0.91 mM to give a buffered-free [Ca2+] of 80 μM. Rates of swelling of mitochondria were determined by differentiation of the A520 time course.14
Deoxyuride-5′-Triphosphate Biotin Nick End Labeling Staining
Left ventricular tissues were fixed in formalin for 24 hours, embedded in paraffin, and sectioned. The apoptotic cells were identified by deoxyuride-5′-triphosphate biotin nick end labeling (TUNEL) using an apoptosis detection kit according to the manufacturer's protocol (Roche Diagnostics, Mannheim, Germany). Twenty photographs (magnification: 400×) of each tissue section were taken by Nikon E400 microscope (Nikon E400, Tokyo, Japan). TUNEL-positive myocytes and the number of total myocytes were counted with Leica Qwin plus V3 software and expressed as the number of TUNEL-positive myocytes per 1,000 myocytes.
Data were expressed as mean ± standard error of the mean, and hemodynamics data were analyzed with two-way analysis of variance (ANOVA) with Tukey post hoc tests (adjusted baseline), when compared with Con group at the same time. For the other data, the Tukey test was used for multiple post hoc comparisons. p < 0.05 (two tailed) was considered statistically significant. All statistical analysis was performed with SPSS 13.0 software (SPSS Inc., Chicago, IL).
LVDP, ±dP/dtmax, heart rate, and coronary artery flow were recorded. At baseline, there were no differences among groups. During reperfusion, LVDP in CsA group was significantly higher than Con group at 5, 15, and 30 minutes. ±dP/dtmax in CsA and Ipo groups were significantly higher than Con group during reperfusion period. There was no significant difference of LVDP and ±dP/dtmax in Ipo group compared with Con group except ±dP/dtmax at 60 minutes of reperfusion. Coronary artery flow and heart rate were decreased at reperfusion in all the groups compared with the baseline. However, there were no significant differences in Ipo and CsA groups compared with Con group. All the data were summarized in Table 1 and Figure 1.
Mitochondrial Permeabilization and Cytochrome C Release
Cytosolic cytochrome C concentrations were higher in Con group compared with Ipo and CsA groups (8.064 ± 0.68 AU vs. 3.24 ± 0.36 AU vs. 3.78 ± 0.57 AU, respectively, p < 0.05) (Figure 2).
Evaluation of mPTP Opening Under Deenergized Conditions
To establish whether the mPTP itself was directly inhibited by classic postconditioning and cyclosporine A postconditioning, mitochondria were rapidly prepared from Con, Ipo, and CsA groups at one of two points during the perfusion protocol: before ischemia and 5 minutes after reperfusion. The extent of mPTP opening was expressed in terms of the initial rate of swelling as described previously.14 Mean data for mitochondria from Con, Ipo, and CsA groups (n = 4) were shown in Figure 3 and confirmed that the decreased sensitivity of mPTP opening to [Ca2+] caused by classic postconditioning and cyclosporine A postconditioning was reproducible and statistically significant. Mean data were also presented for mitochondria isolated from Con, Ipo, and CsA groups before global ischemia, and there were no significant differences among groups.
Effects of Apoptosis on Myocardium
With in situ end labeling of tissue sections, myocardial specimens were examined for final stages of apoptosis. Positive staining was observed in Con, Ipo, and CsA groups (3.60 ± 0.89 vs. 1.38 ± 0.41 vs. 1.10 ± 0.22 per 1,000 nuclei, respectively) after cardioplegic arrest and reperfusion, and there was a significant increase in Con group, compared with Ipo and CsA groups (p < 0.05) (Figure 4).
The data presented in this study showed that both classic postconditioning and cyclosporine A postconditioning could provide protection of postcardioplegic myocardium arrest against reperfusion injury. The mechanism was associated with preventing release of cytochrome C into cytoplasm, inhibiting opening of mPTP, decreasing the rate of apoptosis of myocardium. Feng et al.15 demonstrated postischemic myocardium after cardioplegic arrest experienced apoptotic pathologic process. Cytochrome C was an important component of mitochondrial oxidative phosphorylation. A release of cytochrome C occurring at the onset of mitochondrial dysfunction may be responsible for deficits in mitochondrial respiration. The Western blotting data in our study demonstrated diffusion of cytochrome C from the mitochondria into cytosol owing to permeabilization of the outer mitochondrial membrane. Ipo and CsA hearts had lower levels of cytochrome C than Con hearts in cytosol. It has been suggested that prolongation of acidosis during reperfusion was the determinant for the protective effects of postconditioning.16,17 A recent study demonstrated that an effective length of postconditioning cycles may critically reduce catabolite washout resulting in attenuated transmembrane H+ gradient and decreased activity of Na+/H+ exchanger and Na+/HCO3− symport.18 Low pH of reperfusion could inhibit opening of mPTP,19 explaining why mPTP can be inhibited by postconditioning. During the first 5 minutes of reperfusion, we administered cyclosporine A, a potent inhibitor of mitochondrial permeability transition that seemed to effectively inhibit the opening of mPTP in our study.
Many studies have demonstrated that inhibition of mPTP opening at reperfusion resulted in marked protection against experimental ischemia-reperfusion injury.7,11,20 Argaud et al.7 showed that postconditioning altered the threshold for mPTP opening, so that a greater [Ca2+] load was required to open mPTP in rabbit hearts. In this study, we evaluated the opening of mPTP, using a spectrophotometer under deenergized conditions. These conditions eliminated factors that might have indirect effects on mPTP opening such as changes in membrane potential or calcium transport into, and accumulation within, the mitochondrial matrix. As such, this technique should detect the direct effect of Ipo and CsA on the mPTP mechanism. The result showed that the sensitivity of mPTP opening to [Ca2+] was decreased significantly in Ipo and CsA groups, when compared with Con group. Cyclosporine A probably inhibited the mitochondrial permeability transition by preventing the calcium-induced interaction of cyclophilin D with a pore component21,22 and may play the same role in reperfusion as classic postconditioning. Exactly how these pathways lead to inhibition of the mPTP with consequent protection of the heart remains unclear. They may desensitize the mPTP for [Ca2+], or the activation of some kinases cascade pathways may inhibit mPTP opening.
Evidence for myocardial apoptosis was verified by the increased prevalence of TUNEL-positive nuclei. We found the apoptosis rate was less in Ipo and CsA groups than in Con group. Release of cytochrome C from the intermembrane space into the cytosol is a central event during initiation of apoptosis in response to death stimuli.23 Cyclosporine A can prevent the mitochondrial permeabilization and maintain integrity of the mitochondrial outer membrane, reducing myocardial apoptosis.24,25 Schmitt et al.26 demonstrated that myocardial apoptosis was evident early after ischemia reperfusion and correlated with declining cardiac contractility. We demonstrated cyclosporine A postconditioning hearts had better cardiac function than control hearts, such as in LVDP, ±dP/dtmax, especially, at early reperfusion. However, the classic postconditioning hearts did not show significantly better performance except ±dP/dtmax at 60 minutes of reperfusion. Penna et al.27 also found that postconditioning did not improve the postischemic systolic dysfunction in isolated male rat hearts subjected to 30 minutes of global ischemia and 120 minutes of reperfusion but could prevent the postischemic systolic dysfunction in female rat hearts. In this study, we also used the male Sprague–Dawley rats; gender may have influenced the efficacy of postconditioning. Reperfusion injury could also result in myocardial stunning, which was the postischemic contractile dysfunction in the absence of cell death. Both stunning and apoptosis could lead to contractile dysfunction but may not influence coronary flow. This could explain the phenomenon that coronary flow did not follow the similar recovery to contractile function in this study.
In the isolated perfused heart model, we initially demonstrated protection when cyclosporine A was administered during the first 5 minutes of reperfusion only to cover the crucial time period during which mPTP opening has been demonstrated to occur.5 The effectiveness was the same as in classic postconditioning for preserving the function of mPTP. Although a full demonstration of a causal relationship between postconditioning and inhibition of the mPTP will require further investigation, the present data strongly suggested that mPTP was an important mediator of this cardioprotection. Oka et al.28,29 had used cyclosporine A postconditioning in a neonatal animal model of cardioplegic arrest and demonstrated that cyclosporine A had superior mitochondrial preservation for neonatal animals. However, the drug concentration, duration, and animal age are different from our research.
Postconditioning and cyclosporine A postconditioning have demonstrated beneficial functional effects in patients who underwent coronary angioplasty for acute coronary occlusion.30,31 In the future, we need to confirm whether cyclosporine A postconditioning can have a protective effect in a simulated adult model of cardiac surgery and provide a new myocardial protection strategy for clinical cardiac surgery.
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