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Department of Surgery II, Kochi Medical School, Kohasu, Nankoku, Kochi 7838505, Japan
Submitted 19 February 2003 ; accepted in final form 16 June 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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reactive oxygen species; 3-methyl-1-phenyl-2-pyrazolin-5-one; ion channels; mitochondria; membrane permeability
and OH– and Ca2+ overload (9). Even though endogenous antioxidants such as superoxide dismutase, glutathione peroxidase, and catalase are activated because of the oxidative stress following ischemia, their levels are not high enough to prevent reperfusion injury (5). Hence there is always a need for exogenous supplement of antioxidants, especially after acute ischemia, which has been suggested by many studies, demonstrating the role of exogenous radical scavengers in preventing reperfusion injury by inhibition of reactive oxygen species (ROS) (3, 36) and thus preserving the myocardium. Recent studies revealed that necrotic and apoptotic cell death results after the opening of a latent nonspecific pore in the inner membrane of the mitochondria, known as the mitochondrial permeability transition pore (PTP) (3, 14, 15, 36). The regulation of the PTP plays a key role in maintaining the impermeability of the mitochondrial inner membrane to all but a few selected metabolites, thus helping to maintain the membrane potential, which drives ATP synthesis during oxidative phosphorylation. Crompton (3) demonstrated that increased formation of ROS leads to the opening of the PTP. Recent studies demonstrated that upregulation in expression of Bcl-2, the antiapoptotic protein located in the outer membrane of the mitochondria, was able to inhibit the PTP during reperfusion states (13).
3-Methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) is a potent scavenger of hydroxy radicals that has the ability to inhibit lipoxygenase metabolism (25) and has been demonstrated to be very successful clinically in patients with acute cerebral ischemia (18, 23). Minhaz et al. (22) demonstrated the protective role of MCI-186 in reperfusion injury of the heart, but the exact mechanism by which this agent preserves the myocardium is still not clear.
We demonstrate here the role of MCI-186 in preventing necrosis and pathological apoptosis due to reperfusion injury following ischemia and also establish its PTP-inhibiting property and upregulation of Bcl-2 expression. We used an in vivo rat model of 30-min regional ischemia and 120-min reperfusion and isolated mitochondria along with the well-established PTP openers lonidamine and atractyloside to prove our hypothesis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MCI-186 was a kind gift from Mitsubishi Chemical Industries. The drug was dissolved in NaOH, diluted with distilled water, and pH-adjusted to 7.4 according to the manufacturer's instructions. Lonidamine and atractyloside were purchased from Sigma. Lonidamine was dissolved in glucamine and subsequently diluted to the required concentration before injection. Atractyloside was dissolved in distilled water before injection. All other chemicals were purchased at the highest grade available.
In Vivo Regional Ischemia-Reperfusion
General preparations. The experimental model was similar to the one used in our earlier study (37). In brief, the animals were anesthetized with phenobarbital (30 mg/kg ip), subsequently intubated, and ventilated with a small Harvard rodent ventilator (South Natick, MA) with supplemental O2 set at a tidal volume of 2.0 ml/stroke and at a rate of 70 strokes/min. Heart rate was monitored from ECG tracings and blood pressure was monitored through femoral artery cannulation throughout the study. Animals were systemically heparinized (500 IU/kg body wt) 5 min before left coronary artery (LCA) occlusion in all groups.
Surgical procedures. The chest was opened by a left thoracotomy at the fourth intercostal space. and the ribs were gently spread. The pericardium was carefully dissected and retracted.
The left main coronary artery was identified. A 7-0 prolene suture with an atraumatic needle was inserted 
0.5 mm into the myocardium 2–3 mm away from the origin of the LCA (just beneath the left atrial appendage) after the left auricle was pushed aside with a small noncrushing microforceps. The suture was then connected to the snare and an elastic arch. The snare was then tightened, and coronary artery occlusion was confirmed by ST segment elevation in the ECG and presence of regional cyanosis in the myocardium. When either of these signs was not seen, the LCA was re occluded immediately. Thirty minutes after occlusion, the snare was released and the reperfusion of the myocardium was visually confirmed. The heart was then reperfused for 120 min.
Experimental protocol. The rats were divided into nine groups. All rats were subjected to 30 min of regional ischemia and 120 min of reperfusion. Five rats in each group, used for Western blotting evaluation, were subjected to 12-h reperfusion after 30-min ischemia. In the sham-treated group, rats underwent 30-min ischemia and 120-min reperfusion without any treatment (n = 14). In the MCI-186 group, rats were treated with MCI-186 (10 mg/kg body wt) as an intravenous infusion starting from 30 min before ischemia and continuing up to 10 min of reperfusion (n = 13). In the MCI-186 + lonidamine group, rats were treated with lonidamine (10 mg/kg body wt), a PTP activator, 30 min before MCI-186 infusion and with MCI-186 (10 mg/kg) 30 min before ischemia (n = 12). Previous studies in our laboratory (28) demonstrated that lonidamine at this dose is efficient in activating the PTP. In the MCI-186 + atractyloside group, rats were treated with atractyloside(5 mg/kg) by intravenous infusion 30 min before MCI-186 treatment. MCI-186 was infused in the same dose as above starting from 30 min before ischemia (n = 12). In the allopurinol group, rats were treated with allopurinol (5 mg/kg) by intravenous infusion for 45 min starting from 10 min before ischemia. Allopurinol at this dose has been demonstrated to exert a cardioprotective effect (17). In the allopurinol + lonidamine and allopurinol + atractyloside groups, rats were treated with lonidamine (10 mg/kg body wt) or atractyloside (5 mg/kg body wt) by intravenous infusion for 30 min before allopurinol treatment and were subjected to ischemia-reperfusion. In the last two groups, rats were subjected to ischemia-reperfusion after treatment with lonidamine (10 mg/kg body wt iv) or atractyloside (5 mg/kg body wt iv) without MCI-186 treatment (n = 6 for each group).
Infarct size assessment. At the end of reperfusion, the ligature around the LCA was retightened and 1 ml of 1% Evans blue dye was injected via the left femoral vein to estimate the area perfused by the occluded artery. The animals were immediately killed with an excess dose of phenobarbital, and the heart was removed. The area at risk was determined by negative staining with Evans blue, and the infarcted area was identified as the unstained area within the risk area after 2% triphenyltetrazolium chloride staining. The area of infarcted tissue and the risk zone were determined with IP lab software (version 3; Scanalytics), and the infarct size was expressed as a percentage of the risk area.
Western blotting analysis for Bcl-2. Frozen tissue samples from the risk area after 12-h reperfusion were homogenized in tissue lysis buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.28 U/ml aprotinin, 50 µg/ml leupeptin, and 0.7 µg/ml pepstatin] and clarified by centrifugation at 14,000 g for 20 min at 4°C. Proteins were measured with a bicinchoninic acid kit (Sigma). Equal amounts of protein (50 µg) were run on 15% SDS-polyacrylamide gel. Western blotting was performed as described previously (1) with primary antibody against Bcl-2 (1:1,000, Sigma) and antimouse secondary antibody IgG conjugated with horseradish peroxidase (Cell Signalling Biotechnology). Detection of protein-bound antibody was done with the ECL plus detection system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Immunohistochemistry. The cryostat frozen sections were processed for determination of apoptosis by a single-stranded DNA (ssDNA) method as described previously (10). In brief, the sections, after serial treatment with alcohol, were incubated overnight with ssDNA antibody (Dako Japan), followed by 30-min incubation with the secondary antibody (antirabbit Ig Fab; Dako Japan). Immunohistochemistry was carried out with the ABC staining system (Nichirei) according to the manufacturer's protocol, finally stained with diaminobenzidine solution, and mounted for viewing apoptotic cells. The apoptotic index was collected only in the myocytes, and the prevalence of apoptotic cells was expressed as the percentage of apoptotic cells among the total cardiomyocytes in the ischemic border zone.
Agarose gel electrophoresis of DNA. In a separate series of experiments, the same protocol (n = 5 each) was carried out for the detection of DNA ladder. Transmural myocardial samples from ischemic areas were isolated, frozen in liquid nitrogen, and stored at –80°C. The DNA was extracted from a 70- to 80-mg risk area of the left ventricular myocardium with an apoptotic DNA ladder kit (Roche Diagnostics). The concentration of DNA in each sample was measured by spectrophotometry (260 nm). A similar amount (10 µg) of extracted DNA was loaded on a 1.0% agarose gel electrophoresed on a flatbed gel apparatus (Mupid-3, Advance) at 100 V. The gel was then stained with 0.1% ethidium bromide in Tris-borate EDTA electrophoresis buffer and photographed under transmitted ultraviolet (UV) light.
Myocardial ATP levels. For analysis of myocardial ATP, a left ventricular specimen from the risk area was isolated, frozen in liquid nitrogen, and processed for the analysis of ATP levels as described previously (4, 32). In brief, the frozen samples were crushed and the cells were lysed with Tris · HCl and EDTA buffer, pH 7.4. The lysed sample was then centrifuged, and the supernatant was analyzed for ATP content in a bioluminescence luminometer (LB9501, Lumat) with a commercially available ATP bioluminescence assay kit (Promega).
In Vitro Isolated Mitochondrial Analysis
Mitochondrial isolation. The mitochondria were isolated from left ventricular tissue of normal heart as described previously (30). In brief, a portion of the ventricular tissue was removed, finely minced, and homogenized with a polytron homogenizer in isolation medium (300 mM sucrose, 10 mM MOPS, and 10 mM EGTA, pH 7.35 at 4°C). Extracted mitochondria were suspended in EGTA-free buffer, giving a final protein concentration of 15–20 mg/ml of the suspension buffer. The mitochondrial suspension was stored on ice and used within 4 h of the extraction.
Mitochondrial swelling of MPT and cytochrome c release assay. Mitochondrial swelling was measured in mitochondrial suspensions by the decrease in absorbance at 520 nm (27) with a UV-visible scanning spectrophotometer (Ultrospec3000, Pharmacia Biotech). Mitochondria (0.2 mg protein/ml buffer) were incubated at 25°C in a buffer containing 300 mM sucrose and 10 mM MOPS, pH 7.4 with Tris. After 2min [PDB] , 1mMCa2+, 50 µM lonidamine, or 20 µM atractyloside was added and the absorbance was recorded at 520 nm with or without incubation of the mitochondria with 50 µM MCI-186 for 1 min before addition of the above agents. The optical density for the cytochrome c spectrum was recorded as a reference from 390 to 600 nm.
Statistical Analysis
All results are expressed as means ± SE. The significance of differences between groups in hemodynamics was evaluated by two-way ANOVA. The other parameters were analyzed by one-way ANOVA, followed by the Tukey-Kramer multiple test. A P value of <0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of four rats that died because of serious ventricular fibrillation during the sustained ischemia (3 in the control group and 1 in the MCI-186-treated group treated with lonidamine) were excluded from evaluation.
Hemodynamics
Table 1 summarizes the heart rate, mean arterial blood pressure, and rate-pressure product (RPP) before and after ischemia. Recovery of heart rate was only 72% in the sham group and was significantly increased to 94.1% in the MCI-186-treated group. This effect was completely inhibited on opening the PTP with lonidamine and atractyloside, showing only 75.3% and 73.1% recovery, respectively. Allopurinol treatment also increased the recovery of heart rate (P < 0.05 vs. sham treatment), but opening of the PTP failed to inhibit this protective effect. No significant differences existed between the sham-treated and lonidamine with atractyloside alone-treated groups.
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The RPP in the MCI-186 group recovered to 85.7% compared with only 56.3% in the sham-treated group. Again this recovery was completely inhibited in the lonidamine- and atractyloside-treated groups, showing only 50% and 43.8% recovery, respectively. Allopurinol showed good recovery of RPP with 82.2%; again, there was no effect of PTP opening on this recovery rate.
Infarct Size
Figure 1 shows the infarct size in percentage of area at risk. MCI-186 pretreatment resulted in significant decrease of infarct size from 61.6% of the area at risk in the sham-treated rats to 19.2% of the area at risk, a 50% reduction from the sham-treated rats (P < 0.001).
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Pretreating the rats with lonidamine before prolonged ischemia-reperfusion resulted in significant increase of the infarct size to 57.4% (P < 0.001 vs. MCI-186-treated group), which was the same in the case of atractyloside, showing an infarct area of 58.2% (P < 0.001 vs. MCI-186-treated group). There was no significant difference in infarct size between the sham-treated group and the lonidamine with atractyloside alone-treated group. The PTP openers also failed to inhibit the protective effect afforded by allopurinol.
Myocardial ATP Content
Figure 2 shows the myocardial ATP content among the study groups. The ATP levels in the MCI-186-treated group were significantly higher, with 1.4 = 10–9 mol/g wet wt compared with 0.4 = 10–9 mol/g wet wt in the sham-treated group (P < 0.03). Again, pretreatment with lonidamine markedly reduced the myocardial ATP level to 0.36 = 10–9 mol/g wet wt (P < 0.03 vs. MCI-186-treated group). Treatment with atractyloside before MCI-186 treatment also resulted in low ATP levels (0.5 = 10–9 mol/g wet wt). Allopurinol treatment prevented the myocardial ATP loss, which was continued despite PTP opening. The ATP levels in the lonidamine and atractyloside alone-treated group were not significantly different from the sham-treated group.
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Bcl-2 Expression
Western blotting analysis revealed upregulation in Bcl-2 expression in MCI-186-treated hearts compared with normal or sham-treated hearts, whereas lonidamine pretreatment downregulated Bcl-2 expression, thus suggesting that MCI-186 upregulates Bcl-2 expression (Fig. 3A). Allopurinol failed to demonstrate any upregulation of Bcl-2 expression (Fig. 3A).
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Mitochondrial Swelling
To determine whether MCI-186 prevents the mitochondrial swelling caused by PTP opening, we examined the absorbance of the mitochondria at 520 nm. Figure 4 shows the absorbance among the study groups. Addition of 1 mM Ca2+ produced a drop in absorbance due to increased mitochondrial swelling, which was prevented in a dose-dependent manner by the addition of MCI-186, whereas addition of lonidamine or atractyloside before MCI-186 inhibited this protective effect, showing marked mitochondrial swelling (P < 0.05).
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Cytochrome c Release
Figure 5 shows the cytochrome c spectrum recording among the study groups. Remarkably, addition of Ca2+ resulted in increased release of cytochrome c from the mitochondria, seen as the increased peak at 414 nm (P < 0.05). MCI-186 almost completely inhibited this release, whereas opening the PTP with lonidamine or atractyloside before MCI-186 resulted in prevention of this protective effect of MCI-186, probably because of the PTP-dependent action of MCI-186.
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Prevalence of Apoptosis by ssDNA Staining
The reaction product after incubation with anti-SSDNA antibody was dark brown against the hematoxylin background. In the nonischemic area there were no stained nuclei. In the infarct zone, numerous ovoid, centrally oriented nuclei within myofibers contained the reaction product (Fig. 6A).
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The total cells and the ssDNA-positive cells in the border zone were calculated. The percentage of the ssDNA-positive cells in total cells in each field was then calculated. As expected, MCI-186 treatment reduced the prevalence of apoptotic cells to 2.91% compared with 14.3% in the sham-treated group (P < 0.001).
Opening of the PTP by lonidamine and atractyloside treatment inhibited this effect, reverting the prevalence back to 12.5% and 12.8% (P < 0.001), respectively (Fig. 6B). Again, PTP opening failed to inhibit the protective effect of allopurinol.
DNA Fragmentation
As seen in Fig. 7, DNA fragmentation was very prominent in the control group (lane B), whereas MCI-186 pretreatment prevented this fragmentation, showing very minimal laddering (lane C), consistent with ssDNA staining. In contrast, opening the PTP before MCI-186 treatment resulted in increased fragmentation (Fig. 7, lanes D and E), thereby further confirming the PTP-dependent effect of MCI-186.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ROS, including hydrogen peroxide, superoxide radical, and hydroxy radical, increase on reperfusion following ischemia (6, 11, 12). Ischemia results in inhibition of oxidative phosphorylation and hence decreases ATP levels (16). Ischemia also results in inhibition of the respiratory chain, leading to accumulation of the ubisemiquinone. The sudden influx of oxygen into the anoxic cells during reperfusion induces the formation of oxygen free radicals through an interaction with the ubisemiquinone (29).
These increased ROS result in various consequences responsible for reperfusion injury, such as modification of phospholipids and proteins leading to lipid peroxidation and oxidation of thiol groups (34). Dixon et al. (8) demonstrated that oxidative stress also results in depression of the sarcolemmal Ca2+ pump ATPase and Na+-K+-ATPase activities, leading to decreased Ca2+ efflux and increased Ca2+ influx and thus loading the cells with Ca2+ (6). It has been well shown by Andrew Halestrap and others (3, 14, 16) that this combination of oxidative stress and high calcium levels results in the opening of the PTP (31).
PTP is a nonspecific pore in the inner mitochondrial membrane formed with the critical constituents of both inner membrane proteins, such as adenine nucleotide translocator (ANT) and cyclophilin D, and outer membrane proteins, such as porin (voltage-dependent anion channel) and peripheral benzodiazepine receptors. Pro- and antiapoptotic members of the Bcl-2 family have been demonstrated to be present in the outer membrane and to regulate the PTP during stress. The impermeability of the PTP is thus essential to maintenance of the normal functioning of the cell (33, 26, 29).
MCI-186 has been well shown to exert its cytoprotective effect against cerebral ischemia reperfusion injury by inhibiting OH–-and iron-dependent lipid peroxidations, both of which are essential in mediating reperfusion-induced cell damage (34). Lipid peroxidation results in the activation of arachidonic acid metabolism, and Di Paola et al. (7) demonstrated that metabolites of arachidonic acid cause cytochrome c release from mitochondria by opening the PTP. Hence, we proposed that this MCI-186 could also prevent the opening of the PTP and preserve the ischemia reperfusion-mediated heart injury.
Our data obtained after 30-min ischemia and 120-min reperfusion revealed significant reduction in the area of myocardial infarction with MCI-186 pretreatment compared with sham treatment. Moreover, MCI-186 reduced the prevalence of pathological apoptotic cells, possibly by preventing the lethal opening of the PTP, resulting in inhibition of mitochondrial swelling and hence release of cytochrome c from the intermembrane space of the mitochondria and inhibiting the caspase cascade activation, as evidenced from the isolated mitochondrial studies. The PTP-inhibiting property of MCI-186 was further confirmed by complete inhibition of its cytoprotective effects on opening the PTP with lonidamine or atractyloside before treating the animals with MCI-186.
Lonidamine is an indazole carboxylic acid derivative extensively used as an anticancer agent and has been shown to act on mitochondria by activating the PTP through ANT-mediated mechanisms to induce apoptosis, which can be inhibited by overexpression of Bcl-2 (2). Atractyloside is a derivative from thistles shown to inhibit the oxidative phosphorylation by blocking the adenine nucleotide transfer across the membranes (35). Hence, in our study, we used these PTP activators to prove our hypothesis.
Even though most of the studies have shown elevated ROS levels within a few minutes after the reperfusion, its increase during ischemia still remains controversial (6). Some studies suggested the possibility of accumulation of ROS during ischemia, and this was true in our study, where hearts treated with MCI-186 before ischemia showed marked improvement over hearts treated with the drug just before reperfusion (data not shown).
Jennings et al. (19) reported that when the mitochondria accumulate Ca2+, ATP synthesis ceases, and Crompton (3) and others showed that this failure of ATP generation is preceded by opening of the PTP, thus allowing rapid entry of Ca2+ into the mitochondria. In our study, the MCI-186-treated group had well-maintained myocardial ATP levels after reperfusion, which was inhibited by opening the PTP, resulting in significant loss of ATP levels. Thus it is more likely that MCI-186 prevented PTP mitochondrial pore opening.
According to Martinou et al. (21), the most common mode of cytochrome c release is following the outer mitochondrial membrane rupture as a result of mitochondrial matrix swelling due to the opening of the PTP in conditions of reduced Bcl-2 expression. In the present study, MCI-186 prevented mitochondrial swelling and cytochrome c release, resulting in fewer apoptotic cells. We observed increased expression of Bcl-2 with MCI-186 after 12-h reperfusion. Although at this point we are unable to show the effects of MCI-186 in delayed protection of the myocardium, we believe the upregulation of Bcl-2 expression could play a key role in the delayed protection of the myocardium by MCI-186.
Because elevated ROS opens the PTP (3, 14, 16) it is conceivable that all the radical scavengers exhibit their cytoprotective effects by inhibiting the opening of the PTP as their final step. However, this was not true in our study, where lonidamine or atractyloside pretreatment failed to inhibit the cytoprotective effect of allopurinol, another commonly used potent radical scavenger, and allopurinol pretreatment failed to upregulate Bcl-2 expression, thus suggesting different pathways of radical scavengers in protecting the myocardium.
In summary, our results suggest the following. 1) Pretreatment with the radical scavenger MCI-186 before prolonged ischemia prevents the opening of the PTP by inhibiting the cellular Ca2+ overload due to oxidative stress, thus preventing loss of ATP and reducing necrotic cell death. 2) By inhibiting PTP opening, MCI-186 also prevents mitochondrial swelling and release of cytochrome c, thus reducing the prevalence of pathological apoptosis. Our study also suggested that ROS are formed not only on reperfusion but also during ischemia. Future studies will be required to demonstrate the role of Bcl-2 upregulation in protection with MCI-186 and the exact mechanisms of formation of ROS during ischemia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in rat ventral prostate during postnatal development, after androgen ablation, and after androgen replacement. Endocrinology 139: 3005–3013, 1998.This article has been cited by other articles:
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P < 0.05 vs. MCI-186 + LND or ATR group.
P > 0.05 vs. MCI-186-treated group.
P > 0.05 vs. sham group. B: allopurinol treatment had no effect on Bcl-2 upregulation, which was seen even in the accompanying quantitative evaluation. 
