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Circulation. 1996;93:1177-1184

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(Circulation. 1996;93:1177-1184.)
© 1996 American Heart Association, Inc.

Articles

Direct Evidence That Initial Oxidative Stress Triggered by Preconditioning Contributes to Second Window of Protection by Endogenous Antioxidant Enzyme in Myocytes

Xiaobo Zhou, MD; Xiaolin Zhai, MD; Muhammad Ashraf, PhD

From the Department of Pathology and Laboratory Medicine, University of Cincinnati (Ohio) Medical Center.

Correspondence to Muhammad Ashraf, PhD, Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45267-0529.


*    Abstract
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 *Abstract
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 down arrowMethods
down arrowResults
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Background We tested the hypothesis that late preconditioning is associated with increased antioxidant enzyme activity induced by initial oxidative stress.

Methods and Results Isolated rat myocytes were preconditioned either with two cycles of 5 minutes of anoxia and 5 minutes of reoxygenation or with exogenous superoxide anion (O2-) generated by reaction of xanthine oxidase with xanthine. Myocytes were allowed to recover for 60 minutes or 24 hours, after which they were subjected to 60 minutes of anoxia and 60 minutes of reoxygenation. After 60 minutes or 24 hours, the protection was evidenced by decreased O2- production, increased Mn superoxide dismutase (Mn-SOD) activity, increased cell viability, decreased LDH release, reduced malondialdehyde formation, high-energy phosphate preservation, and improved cell morphology in preconditioned and O2--treated myocytes. Immediately after treatment with O2- or repetitive, brief anoxia, O2- production was increased in myocytes. Longer anoxia resulted in loss of Mn-SOD activity in anoxic controls 24 hours later, whereas it was significantly increased in preconditioned and O2--treated myocytes. O2- production was inhibited in preconditioned and O2--treated myocytes. Myocytes treated with Mn-SOD during short, intermittent anoxia exhibited decreased activity of Mn-SOD and increased O2- production 24 hours later. Mn-SOD activity in late preconditioning was considerably higher than that in classic preconditioning.

Conclusions These results suggest that a burst of oxygen free radicals generated during the initial periods of brief, repetitive anoxia increases myocardial antioxidant activity 24 hours later and that it contributes to the late cardioprotective effect of preconditioning.


Key Words: free radicals • antioxidants • cells • myocardium • hypoxia • ischemia


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowResults
down arrowDiscussion
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Brief episodes of ischemia and reperfusion increase myocardial tolerance to a subsequent sustained period of ischemia.1 2 Myocardial protection associated with classic ischemic preconditioning has been shown to be transient and wanes within 1 to 2 hours.3 Several recent studies4 5 6 7 demonstrated that ischemic preconditioning triggers a late phase of protection 24 hours after the initial preconditioning of hearts. The mechanism of the late phase of preconditioning is unknown; it appears to be different from that of classic ischemic preconditioning. The latter is mediated by adenosine,8 whereas the late effect involves the transcription of mRNA and subsequent synthesis of certain protective proteins, including antioxidant proteins.9 Hoshida et al5 examined antioxidant activity in the preconditioned canine myocardium after 24 hours of sublethal ischemia and found that the reduction of infarct size was associated with the increase in Mn superoxide dismutase (Mn-SOD) activity. It is unclear, however, whether a burst of oxygen free radicals generated immediately after brief, repeated ischemia elicits a compensatory response in the antioxidant reserve of the myocardium 24 hours later.

We therefore tested the hypothesis that the late cardioprotection of preconditioning is associated with an increase in antioxidant enzyme activity induced by the initial oxidative stress. To do this, we directly measured superoxide anion (O2-) production and determined its effect on Mn-SOD contents during preconditioning. Isolated myocytes offer the best experimental model to study specific effects of free radicals on preconditioning, since this model is free of collateral blood flow and noncardiac cells that may influence the amount and types of different radical production, complicating the interpretation of results. Moreover, isolated myocytes are known to produce oxygen free radicals during anoxia and reoxygenation.10 We also tested the hypothesis that exogenous O2- generated by xanthine oxidase and xanthine reaction may precondition myocytes against anoxic injury. The results of this study demonstrate that oxygen free radicals produced during brief, intermittent anoxia may cause increased activity of antioxidant enzymes that appear to be responsible for protection of myocytes against anoxia and reoxygenation injury.


*    Methods
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 up arrowIntroduction
 *Methods
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Reagents
The reagents used in the experiments were as follows: collagenase type II (Worthington Biochemical Corp); laminin (Collaborative Research Inc); medium 199 (Gibco BRL); and xanthine, xanthine oxidase, SOD, hypoxanthine, and cytochrome c (all Sigma).

Animals
All animals received humane care in accordance with Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985).

Isolation of Myocytes
Calcium-tolerant ventricular myocytes were isolated from hearts of adult rats according to previously published procedures.10 11 In brief, male Sprague-Dawley rats weighing 200 to 250 g were anesthetized by injection of 3 mg/100 g pentobarbital IP and anticoagulated with heparin sodium (100 IU/100 g IV). The hearts were rapidly excised and arrested by immersion in cold (4°C) perfusion solution. The hearts were then perfused in the Langendorff mode with low-calcium medium and gassed with 95% O2/5% CO2 at 37°C. After 5 minutes, the hearts were perfused with the same medium plus 0.1% collagenase and 0.1% albumin at a constant flow of 6 mL/min for about 30 minutes, during which calcium was added gradually to the perfusion medium to a maximum final concentration of 1 mmol/L. After 30 minutes, the ventricles were minced and incubated for 10 minutes in fresh medium containing 0.1% collagenase, 0.1% albumin, and 1 mmol/L calcium in a water bath at 37°C. The suspension was filtered through 200-µm nylon mesh and centrifuged slowly. The cell pellet was resuspended in medium 199 containing 1% albumin and 1 mmol/L calcium, and cells were allowed to settle at 37°C. The cells were washed three times with similar medium to remove dead cells. The cell pellet was resuspended in medium 199 containing 10% calf serum, 2 mmol/L glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin. A total of 4x106 to 5x106 rod-shaped cells were obtained from each heart, depending on the collagenase batch. The percentage of viable myocytes (rod-shaped and beating) was >95% in each preparation. Approximately 2.4x105 rod-shaped cells were plated in 60-mm laminin-coated dishes and incubated overnight in a CO2 incubator (95% air/5% CO2). After 24 hours of incubation, the percentage of viable myocytes was {approx}95% in each preparation.

Anoxia and Reoxygenation
Myocytes cultured in medium 199 were transferred into the anoxic chamber (Forma 1025 Anaerobic System) through the gas-interchange cycle. Once inside, the cells were washed three times with anoxic Tyrode's solution containing (mmol/L): NaCl 125, KCl 2.6, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.0, and HEPES 25, pH 7.4, bubbled with 100% N2 at 37°C for 2 hours. It took 2 minutes to wash each dish, and this time was not included in the "anoxic incubation period" calculation. The cells were subjected to anoxic incubation at 37°C with 3 mL anoxic Tyrode's solution per dish. The O2 content of the air inside the chamber and that of the medium was <0.1% during the entire period of the experiment, as measured by the Cavitron/LexO2 CON-KTM DC-60 total O2 content analyzer. This is at the limit of instrument resolution (0.1 vol%). At the end of the anoxia, the dishes were transferred to a CO2 water-jacketed incubator with a humidified atmosphere of 95% air/5% CO2 to begin reoxygenation.

Experimental Protocol
Myocytes were randomly assigned into six groups (Fig 1Down). All groups were performed in six replicates (n=6 per group).



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  		Figure 1. Experimental protocol. Myocytes were preconditioned (PC) with two cycles of 5 minutes of anoxia and 5 minutes of reoxygenation. Superoxide dismutase (SOD) was administered during brief, repetitive anoxia. Myocytes were also preconditioned with exogenous O2- produced by xanthine oxidase and xanthine reaction. The normal control myocytes and the anoxic control myocytes were incubated for 24 hours and 20 minutes, as were all other experimental groups. After 24 hours, myocytes in all groups were subjected to 60 minutes of anoxia and 60 minutes of reoxygenation.

Group 1: Normal control. Myocytes were continuously incubated in aerobic solutions for the duration of experiments.

Group 2: Anoxic control. Myocytes were aerobically incubated for 24 hours and 20 minutes before they were subjected to 60 minutes of anoxia followed by 60 minutes of reoxygenation.

Group 3: Classic preconditioning. Myocytes were preconditioned with two cycles of 5 minutes of anoxia and 5 minutes of reoxygenation. The myocytes were allowed to recover in medium 199 in a CO2 water-jacketed incubator. After 60 minutes of incubation, cells were subjected to 60 minutes of anoxia followed by 60 minutes of reoxygenation.

Group 4: Late preconditioning. Myocytes were preconditioned with two cycles of 5 minutes of anoxia and 5 minutes of reoxygenation. The myocytes were allowed to recover in medium 199 in a CO2 water-jacketed incubator. After 24 hours of incubation, cells were subjected to 60 minutes of anoxia followed by 60 minutes of reoxygenation.

Group 5: Preconditioning and SOD. The protocol was similar to that of group 4, except that an O2- scavenger, SOD (150 U/mL), was added to the Tyrode's solution during the preconditioning period. This dosage was found to be sufficient to quench O2-.12

Group 6: Preconditioning with exogenous O2-. In this group, myocytes were preconditioned with exogenous O2-, ie, two cycles of 5 minutes of incubation with the Tyrode's solution containing 100 µmol/L xanthine and 0.05 U/mL xanthine oxidase13 and 5 minutes of incubation with the Tyrode's solution without xanthine and xanthine oxidase, during which the myocytes were washed several times with xanthine- and xanthine oxidase–free Tyrode's solution to make sure that cells were free of exogenous xanthine and xanthine oxidase. Exogenous O2- was generated by xanthine oxidase acting on xanthine. Addition of either xanthine or xanthine oxidase alone to the Tyrode's solution did not have any effects on myocytes (data not shown).

The supernatant in the culture dishes was collected before and after preconditioning sequences and at the end of reoxygenation after longer anoxia for analysis of O2- and at the end of reoxygenation for LDH. Cells were taken before and after preconditioning sequences and at the end of reoxygenation after longer anoxia for SOD and at the end of reoxygenation for malondialdehyde (MDA), adenine nucleotides, cell viability, and morphological changes. O2- and LDH were measured and cell viability and cell morphology were evaluated immediately after samples were collected. Cells were stored at -70°C for later analysis for MDA and adenine nucleotides.

Measurement of O2-
O2- was quantified spectrophotometrically by monitoring of SOD-inhibitable cytochrome c reduction.14 Briefly, 50 µL of 40 µmol/L cytochrome c and 500 µL of sample were mixed immediately, and 1 mL buffer containing 50 mmol/L potassium phosphate and 0.1 mmol/L EDTA was added to the mixture. In controls, 150 µL of 20-µg/mL SOD was added. The reaction was allowed to proceed for 10 minutes at room temperature, and the absorbance was measured at 550 nm. Reduced cytochrome c was calculated by use of a molar absorption coefficient of 21 000 mol-1·cm-1.15 The results were expressed as nanomoles per milligram protein.

Measurement of SOD Activity
SOD activity was determined by its inhibitory action on the O2--dependent reduction of ferricytochrome by xanthine–xanthine oxidase.16 Briefly, isolated myocytes were homogenized with a Polytron homogenizer in a measured volume of ice-cold Tris-sucrose buffer containing 0.25 mol/L sucrose, 10 mmol/L Tris-HCl, 1 mmol/L EDTA, and 0.5 mmol/L dithiothreitol, pH 7.5. After centrifugation at 1000g for 15 minutes, the supernatant was centrifuged at 10 000g for 20 minutes to obtain the mitochondrial pellet that was used as a source of Mn-SOD. The supernatant was again centrifuged at 105 000g for 1 hour. The supernatant was used to assay Cu/Zn SOD. The final concentrations in the assay medium (total volume, 1 mL) were 100 mmol/L cytochrome c, 100 mmol/L hypoxanthine, 10 mmol/L Tris-HCl, and 50 to 80 µg enzyme protein. The reaction was initiated by addition of 8 mU xanthine oxidase. The inhibition rate of cytochrome c reduction was calculated. SOD activity was determined from the standard curve of percentage of inhibition–SOD activity. The curve was obtained by running with commercial standard SOD. SOD activity was expressed as units per milligram protein.

Measurement of Myocyte Protein Content
Protein content was measured by the method described previously.10 11 The method we used is from the Sigma assay kit. This method is based on the fact that brilliant blue G reacts with protein in an acid-alcohol medium to form a blue protein dye complex. The color, measured at 595 nm, is proportional to the protein concentration. Protein content and other parameters were measured from separate dishes containing identical numbers of myocytes from one heart preparation. The myocytes were mixed with 2 mL of 6% cold perchloric acid. The cell suspension was heated at 70°C for 20 minutes and then centrifuged at an acceleration of 1000g for 20 minutes. The pellet was dissolved in 2 mL of 0.1N NaOH solution and was cooled in ice for 20 minutes, after which it was centrifuged again at 1000g for 20 minutes. The absorbance of supernatant was measured by brilliant blue G (Coomassie blue) reaction with protein. For standards, 300 µg/mL BSA fraction V, dissolved in 0.1N NaOH, was used, and the absorbance was measured at 595 nm. The protein contents of each dish ranged from 1.0 to 1.2 mg. Using the protein content measured, we normalized all the data in the present study.

Measurement of LDH
Supernatant (1 mL) was collected for determination of LDH at the end of reoxygenation after longer anoxia. Spectrophotometric enzyme assay was performed with a Sigma assay kit. Measurement of enzyme activity was based on the oxidation of lactate and the rate of increase in absorbance at 340 nm. The activity of LDH was expressed as units per milligram protein.

Measurement of High-Energy Phosphate
Adenine nucleotides were assayed with high-performance liquid chromatography (HPLC) analysis.17 18 Isolated myocytes were homogenized with a Polytron homogenizer in 1 mL of 0.3N perchloric acid. The extracts were neutralized with KOH and filtered. Twenty microliters of the extract was injected into the reverse-phase HPLC unit (model 110B, Beckman Instruments Inc). The mobile phase was 240 mL acetonitrile plus distilled water to a final volume of 1100 mL containing 0.65% KH2PO4 and 0.3% tetrabutylammonium phosphate (pH 5.8). Flow rate was 1.0 mL/min on a Hibar RT column (Lichrosorb-RP-18, 10 µm, 25 cm x 4 mm). The detector was set at a wavelength of 254 nm coupled with an IBM-PC. The concentration of adenine nucleotides was expressed as nanomoles per milligram protein. The following intermediates were measured: ATP, ADP, and AMP. The values obtained were used to calculate the following indexes of myocardial energy status: Total adenine nucleotides=ATP+ADP+AMP; energy charge=(ATP+0.5ADP)/(ATP+ADP+AMP).

Measurement of Lipid Peroxidation
Lipid peroxidation was determined by the measurement of MDA in the isolated myocytes. MDA was measured with the thiobarbituric acid reaction, which reveals several oxidized substances, including MDA, and has been used frequently by many investigators to measure MDA as an index of lipid peroxidation.19 Myocytes were homogenized in 9 vol 1.15% KCl solution at 4°C. To prevent auto-oxidation of the samples, 0.05% butylated hydroxytoluene was added to the homogenate, and the solution was bubbled with N2. To 0.1 mL of the homogenate, 0.4 mL of 8.1% sodium dodecyl sulfate, 3 mL of 20% acetic acid, and 3 mL of 0.8% thiobarbituric acid were added, and it was incubated for 90 minutes at 95°C. After cooling, 1 mL of distilled water was added, and the sample was extracted with 5 mL of butanol/pyridine (15:1). The optical density of the upper organic layer was read at 532 nm with a Beckman DV-40 spectrophotometer. Standard MDA was prepared by acid hydrolysis of MDA-bis dimethyl acetal. The amount of MDA was expressed as nanomoles per milligram protein.

Evaluation of Cell Viability and Cell Morphology
A small aliquot of 0.1% trypan blue was added to the dishes containing myocytes, and after 3 minutes the cells were viewed under a phase-contrast microscope. Dead cells were permeable to trypan blue, whereas viable cells did not take up the dye. By counting of 100 cells (dyed and nondyed), the percentage of viable cells was calculated. Myocytes were examined under a phase-contrast microscope for morphological changes.11

Statistical Analysis
Statistical analysis was based on the guidelines described by Wallenstein et al.20 All data were expressed as mean±SEM. A one-way ANOVA was first carried out to test for any differences between the mean values within the same study. When a significant F value was obtained, comparisons between individual means of groups were performed by Student-Newman-Keuls test. A difference of P<.05 was considered significant.


*    Results
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*Results
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Amount of Cytochrome c Reduction
Fig 2Down demonstrates SOD-inhibitable cytochrome c reduction in different groups. SOD-inhibitable cytochrome c reduction represents O2- generated during anoxia and reoxygenation. Baseline values were similar among groups. Immediately after brief, intermittent anoxia, cytochrome c reduction was significantly higher in the classically preconditioned (1.85±0.07 nmol/mg protein), late preconditioned (1.92±0.08 nmol/mg protein), and exogenous O2--treated myocytes (1.87±0.08 nmol/mg protein) than in the anoxic control (0.45±0.05 nmol/mg protein, P<.05). SOD treatment abolished the amount of cytochrome c reduction (0.46±0.05 nmol/mg protein). After 60 minutes, cytochrome c reduction after 60 minutes of anoxia and 60 minutes of reoxygenation decreased significantly in the classically preconditioned myocytes (0.58±0.07 nmol/mg protein). After 24 hours, the amount of cytochrome c reduction after 60 minutes of anoxia and 60 minutes of reoxygenation decreased significantly in the late preconditioned (0.62±0.06 nmol/mg protein) and exogenous O2--treated (0.56±0.05 nmol/mg protein) myocytes than in the anoxic control (3.16±0.09 nmol/mg protein, P<.05). There was a significant increase of cytochrome c reduction in the myocytes treated with SOD (3.71±0.11 nmol/mg protein).



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  		Figure 2. Graph shows superoxide dismutase (SOD)-inhibitable cytochrome c reduction, which represents O2- production during anoxia and reoxygenation in different groups. Baseline and preconditioning (PC) readings were made before and after brief preconditioning sequences. Reoxygenation readings were made at the end of 60 minutes of reoxygenation after 60 minutes of anoxia. *P<.05 vs anoxic control group. n=6 per group.

SOD Activity
Fig 3Down shows the Mn-SOD activity in different groups. No difference in Mn-SOD activity was observed in baseline parameters among groups. Similarly, there was no difference in Mn-SOD activity immediately after preconditioning sequences among groups. Mn-SOD activity after 60 minutes of anoxia and 60 minutes of reoxygenation increased significantly in the classically preconditioned myocytes (1.57±0.08 U/mg protein). Twenty-four hours later, longer anoxia resulted in reduced activity of Mn-SOD in anoxic control (0.71±0.06 U/mg protein, P<.05 versus normal control), whereas activity of Mn-SOD increased significantly in the late preconditioned (3.35±0.10 U/mg protein) and O2--treated (3.41±0.12 U/mg protein, P<.05 versus anoxic control) myocytes. On the contrary, Mn-SOD activity was reduced in the myocytes treated with SOD during short, repetitive anoxia (0.62±0.05 U/mg protein, P<.05 versus normal control). Mn-SOD activity in late preconditioning was considerably higher than that in classic preconditioning. There was no difference in Cu/Zn SOD activity among groups in baseline, after preconditioning sequences, and after longer anoxia (data not shown).



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  		Figure 3. Graph represents the time course of changes of Mn-superoxide dismutase (SOD) activity in different groups. Baseline and preconditioning (PC) readings were made before and after brief preconditioning sequences. Reoxygenation readings were made at the end of 60 minutes of reoxygenation after 60 minutes of anoxia. *P<.05 vs anoxic control group, {dagger}P<.05 vs normal control, #P<.05 vs late preconditioning and preconditioning with exogenous O2-. n=6 per group.

Cell Viability
Cell viability was determined by trypan blue exclusion. Fig 4Down depicts cell viability in different groups. After 24 hours, when myocytes were exposed to 60 minutes of anoxia followed by 60 minutes of reoxygenation, the percentage of viable cells declined to 29.6±2.5% in the anoxic control group. When myocytes were preconditioned by brief, repeated anoxia or exogenous O2-, the survival rate of myocytes increased significantly in the classically preconditioned (70.4±2.8%), late preconditioned (61.8±3.4%), and O2--treated (63.4±3.5%, P<.05 versus anoxic control) groups. On the contrary, the beneficial effect of preconditioning on the survival rate of myocytes was completely blocked by SOD treatment (32.7±2.2%).



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  		Figure 4. Cell viability was measured at the end of 60 minutes of reoxygenation after 60 minutes of anoxia. SOD indicates superoxide dismutase; PC, preconditioning. *P<.05 vs anoxic control group. n=6 per group.

LDH Leakage
LDH release measured at 60 minutes of reoxygenation after 60 minutes of anoxia is illustrated in Fig 5Down. LDH release was 0.517±0.037 U/mg protein in the anoxic control group and was markedly reduced from myocytes subjected to classic preconditioning, late preconditioning, and preconditioning with exogenous O2- (0.216±0.029, 0.237±0.026, and 0.221±0.032 U/mg protein, respectively, P<.05 versus anoxic control). Treatment with SOD completely blocked the protective effect of anoxic preconditioning on LDH release (0.495±0.021 U/mg protein, P>.05 versus anoxic control).



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  		Figure 5. LDH was measured at the end of 60 minutes of reoxygenation after 60 minutes of anoxia. SOD indicates superoxide dismutase; PC, preconditioning. *P<.05 vs anoxic control group. n=6 per group.

MDA Formation
Twenty-four hours later, MDA formation measured at 60 minutes of reoxygenation after 60 minutes of anoxia is illustrated in Fig 6Down. MDA formation was 0.743±0.048 nmol/mg protein in the anoxic control group and was significantly reduced by classic preconditioning, late preconditioning, and preconditioning with exogenous O2- (0.372±0.033, 0.395±0.038, and 0.426±0.064 nmol/mg protein, respectively, P<.05 versus anoxic control). Treatment with SOD completely abolished the reduction of MDA formation by anoxic preconditioning (0.723±0.026 nmol/mg protein, P>.05 versus anoxic control).



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  		Figure 6. Malondialdehyde (MDA) was measured at the end of 60 minutes of reoxygenation after 60 minutes of anoxia. SOD indicates superoxide dismutase; PC, preconditioning. *P<.05 vs anoxic control group. n=6 per group.

Adenine Nucleotide Contents
The cellular contents of adenine nucleotides at 60 minutes of reoxygenation after 60 minutes of anoxia are summarized in the TableDown. When myocytes were exposed to 60 minutes of anoxia followed by 60 minutes of reoxygenation 60 minutes or 24 hours later, cells preconditioned either by brief, repeated anoxia or by exogenous O2- had significantly higher contents of ATP and total adenine nucleotides, lower ADP and AMP, and higher values of energy charge compared with anoxic control. SOD completely abolished preservation of ATP, total adenine nucleotides, and energy charge observed during anoxic preconditioning.


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  		Table 1. Adenine Nucleotide Contents of Myocytes in Experimental Groups

Cell Morphology
Light photographs of rat myocytes were taken with a phase-contrast microscope (magnification x200) in the presence of trypan blue at the end of 60 minutes of reoxygenation after 60 minutes of anoxia (Fig 7Down). The myocytes in the normal control group maintained their elongated rod-shaped forms and excluded trypan blue stain. After 60 minutes of anoxia and 60 minutes of reoxygenation, many cells in anoxic controls were rounded. Anoxic preconditioning preserved the typical rod-shaped pattern of myocytes, and the number of round myocytes was significantly reduced. The beneficial effect of preconditioning was not observed in the presence of SOD during repeated transient anoxia. Treatment of myocytes with exogenous O2- mimicked anoxic preconditioning in the numbers of viable and nonviable cells.



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  		Figure 7. Photomicrographs (magnification x200) were taken in the presence of trypan blue at the end of 60 minutes of reoxygenation after 60 minutes of anoxia. a, Normal control: Most cells were elongated rod-shaped forms that excluded trypan blue stain. b, Anoxic control: Many cells were rounded and stained positively. c, Classic preconditioning significantly reduced the number of round and stained myocytes. d, Late preconditioning significantly reduced the number of round and stained myocytes. e, Preconditioning plus superoxide dismutase (SOD): The number of rod-shaped cells was decreased, suggesting that the beneficial effect of preconditioning disappeared in the presence of SOD during repetitive, transient anoxia. f, Preconditioning of myocytes with exogenous O2- mimicked anoxic preconditioning.


*    Discussion
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*Discussion
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It was recently reported that the effect of ischemic preconditioning is lost within 2 hours and reappears after 24 hours of brief, repetitive ischemia and reperfusion.4 5 6 7 This phenomenon is called the delayed or late phase of preconditioning. The mechanism for this delayed protection is currently unknown. It may share the same mechanism as that in early preconditioning, or it may have a different signaling pathway. The primary objective of the present study was to test the hypothesis that a burst of oxygen free radical generation immediately after brief, repeated ischemia triggers alteration of antioxidant activity 24 hours later, which accounts for the delayed protection. The results reported here clearly demonstrate that short episodes of anoxia and reoxygenation can precondition myocytes against subsequent sustained anoxic injury 24 hours later; this preconditioning effect is perhaps mediated by a mild burst of oxygen free radicals during intermittent anoxia.

Isolated Myocytes as a Model of Preconditioning Study
Isolated myocytes can readily be obtained as a homogeneous population with little or no contamination by other cell types, and they are maintained in a defined environment that can be readily manipulated. Isolated myocytes were chosen for preconditioning in this initial study because they may provide an ideal experimental model for studying the basic mechanisms of preconditioning at the cellular level independent of interactions with other cell types, which may be helpful in extending our insight into the true nature of preconditioning. One of the first applications of isolated myocytes as a model for a preconditioning study was from Dr Ganote's laboratory. Those investigators used an isolated cardiomyocyte pellet to simulate ischemia. Using this model, they successfully studied the roles of adenosine,21 adenosine A1/A3 receptors,22 protein kinase C,21 and ATP-sensitive potassium channels23 in classic ischemic preconditioning. Another study also showed that preconditioning can be induced in human cardiomyocytes with simulated ischemia.24 However, we cannot rule out the possibility that the isolated myocytes are different from those in the intact hearts in their behavior. Therefore, we cannot exclude the possibility that the finding in this isolated myocyte model is a phenomenon unrelated to that in the intact hearts.

Myocytes Can Be Preconditioned by Transient Anoxia 24 Hours Later
Brief episodes of hypoxia or anoxia have been shown to induce protection from ischemic or anoxic injury in both intact hearts and isolated myocytes. A recent study showed that 5 minutes of hypoxic perfusion of hearts was as effective in inducing protection as 5 minutes of zero- or low-flow ischemia.25 This suggests that the intracellular metabolic consequence of impairment of oxygen delivery triggers preconditioning. In the present study, two cycles of 5 minutes of anoxia followed by 5 minutes of reoxygenation preconditioned myocytes against a subsequent sustained anoxia-induced injury 24 hours later. This protection was associated with decreased O2- production, increased Mn-SOD activity, less cellular enzyme release, preserved high-energy phosphate, decreased lipid peroxidation, increased cell viability, and well-preserved cell morphology. The present study provides direct evidence that preconditioning can protect myocytes against anoxia and reoxygenation injury at the cell level.

Delayed Preconditioning Mediated by Oxygen Free Radicals
It has been reported that delayed ischemic preconditioning reduces infarct size.4 5 6 7 The delayed preconditioning definitely lasts longer than the early phase of preconditioning, although the duration of the delayed protection is not determined yet. This characteristic of delayed preconditioning makes it more important in clinical settings. The subcellular mechanisms, however, must be elucidated before this protection can be manipulated to develop new treatments of acute myocardial infarction.

It is rather controversial whether free radicals mediate the early phase of preconditioning.26 27 28 The present study has shown that early preconditioning is at least in part mediated by initial oxidative stress triggered by short periods of anoxia in this model. The same stimuli are capable of producing both classic and late preconditioning, indicating that preconditioning, regardless of early or late, might share to some degree the same mechanism in rat myocytes. The difference between these two preconditionings could be that initial oxidative stress increases enzyme activity in the early phase of preconditioning through the conformational change in Mn-SOD29 and induces de novo Mn-SOD synthesis in the late phase of preconditioning. This is why the Mn-SOD activity in late-preconditioned myocytes was much higher than that in early preconditioning. On the other hand, it is interesting to note that the protection is nearly identical, whereas Mn-SOD activity is much higher in late than in classic preconditioning. The exact reason for this discrepancy is somewhat unclear. However, it is likely that the amount of Mn-SOD activity in classic preconditioning is already high enough to scavenge O2- produced during 60 minutes of anoxia to the baseline level. Thus, additional amounts of Mn-SOD synthesized in late preconditioning could not further reduce O2- to a level under the baseline. It also appears that during early preconditioning, in addition to increased Mn-SOD activity, there is a possibility of involvement of some unknown cardioprotective proteins, such as heat-shock proteins, which are known to be expressed in myocardial preconditioning.6 9 The major benefit of late preconditioning may be that increased synthesis of various antioxidants and heat-shock proteins causes the cardioprotection to last longer in late than in early preconditioning.

Several lines of evidence4 5 6 from in vivo studies show that the delayed phase of preconditioning involves augmentation of various protective proteins, including heat-shock proteins and antioxidant proteins, especially Mn-SOD. Kuzuya et al4 and Hoshida et al5 reported that preconditioning with brief, repeated ischemia significantly decreased infarct size after a 90-minute occlusion performed 24 hours after preconditioning. This protective effect was associated with a significant increase in myocardial Mn-SOD, glutathione peroxidase, and reductase. This evidence is further supported by the findings of this study that O2- generated by brief, repeated anoxia induces increased activity of Mn-SOD adequate to quench O2- formation at a latter stage if the cells were subjected to prolonged anoxia. The removal of O2- in the chain reaction of deleterious oxygen reactive species further prevented lipid peroxidation and reduced enzyme release. When myocytes were treated with exogenous SOD during the initial preconditioning phase, the beneficial effects were totally abolished. Another interesting finding from this study is that pretreatment with exogenous O2- mimicked brief, repeated anoxia to trigger the delayed protection of preconditioning, which was also associated with increased myocardial Mn-SOD activity. An increase in intracellular Mn-SOD could protect myocytes against extracellular oxygen free radicals, since these small molecular radicals can move in and out of cells freely.30 Myocytes, endothelial cells, and neutrophils are able to generate free radicals, which can move out of cells to attack other targets.31 Thus, antioxidants or antioxidant enzymes can readily quench these oxygen radical species at their generating sites or during their movement to attack other targets in the cells.

Taken together, all of these data provide strong evidence that it is the brief burst of O2- production during short, repetitive anoxia that activates the subcellular machinery leading to activation of myocardial Mn-SOD, an important factor in the protection elicited by preconditioning.

Alternative Mechanisms for the Delayed Preconditioning
An alternative mechanism for the delayed preconditioning may involve increased levels of heat-shock proteins. A variety of stressful stimuli, including hyperthermia, ischemia, and anoxia, induce the synthesis of proteins, leading to the limitation of infarct size. In this scenario, the delayed preconditioning might induce the transcription of mRNA and subsequent synthesis of one or more heat-shock proteins that would then protect the myocardium during sustained ischemia and reperfusion. A study by Marber et al6 supports the concept of a "second window" of preconditioning that was associated with induction of heat-shock proteins. The latter investigators observed that elevation of myocardial heat-shock protein 72 at 24 hours after brief ischemia or heat stress was associated with resistance to myocardial infarction in rabbits. Although we did not examine whether heat-shock proteins were induced by short anoxia in this experimental model, such preconditioning has been shown to induce the expression of heat-shock proteins and antioxidant proteins.9 26 Induction of heat-shock proteins or antioxidative proteins by short preconditioning sequences may be regulated by separate pathways. On the other hand, myocardial ischemia has been shown to induce heat-shock proteins possibly via accumulation of reactive oxygen free radicals such as O2-.32 33 34 Therefore, it seems reasonable to state that the induction of heat-shock proteins may be a part of overall cellular defense mechanisms against the oxidant stress.9 15

Another explanation for the delayed preconditioning relates to the adenosine hypothesis recently proposed by Baxter et al.35 Those investigators have provided evidence that the delayed protection elicited by pretreatment with an adenosine A1 receptor agonist was inhibited by treatment during preconditioning with an adenosine receptor antagonist, showing a common mechanism of classic preconditioning. However, adenosine A1 receptor does not appear to be related to the mechanism proposed in the present study, since adenosine may inhibit O2- production,36 and a recent study in the conscious pig demonstrated that a sequence of brief coronary occlusions activates an unknown endogenous cardioprotective mechanism not mediated by adenosine receptors, which increased the resistance of the myocardium to stunning 24 hours later.37 It is more likely that the ATP-sensitive potassium channel is involved in this phenomenon, since exogenous free radicals have been shown to open this ion channel.38 The ATP-sensitive potassium channel has been shown to be an important mediator of classic preconditioning in intact dog hearts39 40 and in isolated rabbit cardiomyocytes.23 To the best of our knowledge, however, no currently available data show that free radicals can directly activate the ATP-sensitive potassium channel during late preconditioning.

Conclusions
The present results indicate that oxygen free radicals produced during brief, repetitive anoxia sequences increase activity of antioxidant enzymes that appear to be responsible for the late protection of myocytes against anoxia and reoxygenation injury.


*    Acknowledgments
 
This work was supported by National Institutes of Health research grant HL-23597.

Received July 17, 1995; revision received October 20, 1995; accepted October 23, 1995.


*    References
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