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,1
* Department of Medical and Occupational Sciences, Laboratory of Molecular Biology, University of Foggia; and
Department of Emergency and Transplantation, and
Department of Pharmacology and Human Physiology, University of Bari, Italy
2Correspondence: Department of Medical and Occupational Sciences, University of Foggia, Viale Pinto, 71100 Foggia, Italy. E-mail: g.vendemiale{at}unifg.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: hypoxic reperfusion • ischemia-reperfusion • mitochondria • oxidative stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The imbalance between oxygen supply and metabolic demand leads to functional, metabolic, electrophysiological, and morphological alterations, eventually causing cellular death. Reperfusion injury to the myocardium results at least in part from oxygen free radicals (ROS) released from the ischemic tissue upon deoxygenation. This damage may be or may not be reversible, depending on the severity and duration of the ischemic period (2)
. Reversible damage is caused by combinations of free radical attack and transient calcium overload (3)
; irreversible injury occurs when the ischemic period is extended and severe; it is also mediated by oxidative stress and results in myocardial cell death through necrosis or apoptosis (4)
.
Ischemic preconditioning was introduced as a potent form of cardioprotection against ischemic-reperfusion injury, reducing the incidence of postischemic arrhythmias, enhancing the recovery of cardiac function, and reducing the infarct size after global ischemia (5
, 6)
.
Recently, Zhao et al. (7)
have reported that a short series of repetitive cycles of brief reperfusion and reocclusion of the coronary artery applied immediately at the onset of reperfusion, termed "postconditioning," was as effective as preconditioning. They showed that the mechanisms involved in postconditioning protection take place within the first minutes of reperfusion.
Based on these considerations, Kin et al. (8)
recently focused on events in the first minute of reperfusion, reporting that protection was achieved by a 1 min reperfusion modification protocol consisting of three cycles of 10 s each of unrestricted reperfusion, followed by 10 s of reocclusion of the same coronary artery subjected to the occlusion causing the ischemia. The authors agreed that the efficacy of the preconditioning most likely relates to the burst of free radical generation very early during reperfusion; this agrees with Ambrosio’s study, which first demonstrated that a peak of free radical formation is formed early after reperfusion, no later than after 10–20 s of reoxygenation (9)
. Although Zin and Zhao drew attention to free radical production, they did not demonstrate where free radicals are produced or what is the interplay between the generation of ROS and endogenous scavenging mechanisms to effectively neutralize ROS and prevent irreversible cell injury.
The tripeptide glutathione (GSH) is the main intracellular nonenzymatic antioxidant agent. Under oxidative stress, GSH reacts either as an electron donor to neutralize hydrogen peroxides and lipoperoxides or as a direct oxygen free radical scavenger; this results in its depletion and excess of oxidized glutathione (GSSG). Because of its important antioxidant properties, GSH is known to play a pivotal role in myocardial protection against ischemia/reperfusion (I/R) (10)
. Accordingly, we designed a model of isolated and perfused heart, where a short period (3 min) of hypoxic reperfusion was applied immediately at the onset of reperfusion compared with classical normoxic reperfusion to explore the role played by mitochondria in ROS generation as well as mitochondria and tissue redox modifications induced by ischemia-reperfusion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Briefly, male Wistar rats weighing 250–300 g. were heparinized and anesthetized with i.p. Thiopental. Heart were excised and perfused at 37°C under 10 mL/min constant flow. Krebs-Henseleit was used as perfusion buffer, saturated with O2/CO2 (95/5) gas mixture. pO2 was monitored by electrodes (Instech Dual Oxygen, Plymouth Meeting, PA, USA).
Left ventricular (LV) isovolumic pressure was recorded by a strain gauge pressure transducer (Hewlett-Packard Medical Electronic Division, Model 1280C, Waltham, MA, USA). End-diastolic pressure was adjusted to 
5–10 mmHg. Aortic root pressure was monitored by a strength strain gauge pressure transducer (Hewlett-Packard Medical Electronic Division, Model 1280C) via a side arm of the cannula. Pacing electrodes were attached to atrial appendages and the heart was paced at 300 beats/min.
A group of eight hearts were simply perfused and used as preischemic control (group A). In the other groups, 15 min after heart stabilization, hearts were subjected to global ischemia for 45 min by turning off the perfusion system and injecting 4 mL of Saint Thomas II cardioplegic solution within 2 min at 4°C. At the end of 45 min of cardioplegic arrest, a group of eight hearts was reperfused with a Krebs-Henseleit solution buffered with the preischemic pO2 level (pO2 600 mmHg) (normoxic group B). In another group of eight rats, a second reservoir containing Krebs-Henseleit solution (in which O2 and CO2 had not been bubbled at all) was connected to the pump during the first 3 min of reperfusion in order to maintain the solution at the atmospheric pO2 level of 150 mmHg (hypoxic group C). In the absence of hemoglobin, a pO2 level of 600 and 150 mmHg is considered normoxic and hypoxic, respectively, as previously reported (12
, 13)
. Krebs-Henseleit pH was maintained between 7.35 and 7.45. In the hypoxic group 3 min after reperfusion, the pump was shifted to 600 mmHg pO2 mixture to continue a normoxic reperfusion (Fig. 1
). In two groups of 12 hearts the experiment was stopped 3 min after reperfusion in normoxic (group D) and hypoxic rats (group E). Left ventricular pressure curves were recorded just before ischemia and at minutes of reperfusion 1, 2, 3, 4, 5, 10, 20, 30, and 40. The following parameters were measured: left ventricular end-diastolic pressure (LVEDp; mmHg); left ventricular systolic pressure (LVSp; mmHg); left ventricular developed pressure (LVDp=LVSp–LVEDp; mmHg); dP/dt (expressed in terms of percent change over the preischemia value); root aortic pressure (RAp; mmHg).
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Mitochondria isolation, redox measurement, and Western blot analysis
Heart mitochondria were isolated by differential centrifugation as described (14)
. GSH and GSSG levels of mitochondria and heart homogenate were measured (15)
. Total protein concentration was determined by the Lowry micromethod kit (Sigma-Aldrich, St. Louis, MO, USA). Mitochondria protein carbonyl measurement was performed according to Levine et al. (16)
. Qualitative analysis of mitochondria oxidized proteins was performed by Western blot analysis using Oxyblot kit (Chemicon International, El Segundo, CA, USA). Briefly, the same amounts of mitocondrial proteins (15 µg) was reacted with dinitrophenylhydrazine (DNPH) for 20 min, followed by neutralization with a solution containing glicerol and 2-mercaptoethanol, resolved in 12.5% SDS-PAGE, transferred to a nitrocellulose membrane, blocked with nonfat milk, then incubated with a rabbit anti-DNPH antibody as the primary antibody (1:150) at 4°C overnight. After washing, the membrane was incubated with the secondary antibody (1:300) conjugated to horseradish peroxidase (HRP) and detected by a chemiluminescence detection kit (Cell Signaling Technology, Beverly, MA, USA; #7071). Reactive bands were visualized by the enhanced chemiluminescence method on VersaDoc Image System (Bio-Rad, Hercules, CA, USA). Band density was determined with TotalLab software.
Measurement of peroxide production in heart mitochondria
The rate of peroxide production in heart mitochondria was determined by a modification of the method described by Barja as previously reported (15)
. Mitochondria were incubated at 37°C with 5 mM pyruvate plus 2.5 mM malate or with 10 mM succinate in 2 mL of 5 mM phosphate buffer, pH 7.4, containing 0.1 mM EGTA, 3 mM MgCl2, 145 mM KCl, 30 mM HEPES, 0.1 mM homovanilic acid, and 6 U/mL HRP. The incubation was stopped at 5, 10, and 15 min with 1 mL of cold 2 M glycine buffer containing 50 mM EDTA and 2.2 M NaOH. The fluorescence of supernatants was measured using 312 nm as excitation wavelength and 420 nm as emission wavelength. The rate of peroxide production was calculated using a standard curve of H2O2.
Statistical analysis
Data were expressed as mean ± standard deviation of the mean (SD). Since the redox data were not paired, differences between means were analyzed by 1-way ANOVA after Gaussian distribution evaluation by Kolgomorov-Smirnov test. The Tukey Multiple Comparisons Test for all pairs of columns was applied as Post Test. Cardiac function parameters were analyzed by two way ANOVA for repeated measures and Bonferroni correction test as post hoc test.
In all instances, P < 0.05 was taken as the lowest level of significance. The package (GraphPad Prism Software Inc., San Diego, CA, USA) was used to perform all the statistical analyses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Fig. 2
. Baseline left ventricular end-diastolic pressure (LVEDp) was similar in the two groups (5.62±1.061 mmHg vs. 6.25±1.282 mmHg in normoxic and hypoxic group, respectively). Normoxic reperfusion induced a significant decrease of left ventricular development pressure (LVDp) (Fig. 2A
) immediately after reperfusion commenced, which was prevented by hypoxic reperfusion. Normoxic reperfusion induced a significant increase in LVEDp that steeply increased 1 min after reperfusion began; by contrast, LVEDp increase was smaller in the hypoxic group at all times (Fig. 2B
). No difference was found for dP/dt+, dP/dt–, or left ventricular systolic pressure between the two groups. Root aortic pressure was monitored during preischemia and reperfusion time; an increase was recorded after ischemia although no difference was found in the two groups (Table 1)
.
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Hypoxic reperfusion partially prevented myocardial and mitochondrial glutathione depletion and mitochondrial protein oxidation during ischemia-reperfusion
GSH and GSSG levels were measured in heart homogenate and mitochondria from control, normoxic, and hypoxic rats at the end of reperfusion. Heart homogenate GSH levels were significantly lower and GSSG significantly higher in normoxic rats at the end of reperfusion compared with sham-operated rats (P<0.05, Tukey-Kramer test). This effect was completely reverted by hypoxic reperfusion (Fig. 3
). The same changes were found at mitochondrial level (P<0.05 Tukey-Kramer test) (Fig. 4
). Namely, normoxic reperfusion caused a significant increase of GSSG (Fig. 4A
) and a fall of GSH (Fig. 4B
); again, this was prevented by hypoxic reperfusion. In addition, oxidative alterations of mitochondrial proteins were observed after ischemia-reperfusion injury (Fig. 5
): mitochondria from normoxic hearts exhibited a significant increase of protein carbonyls compared with controls (P<0.001, Tukey-Kramer test). Hypoxic reperfusion partially prevented the protein oxidation observed in normoxic hearts (P<0.001; Tukey-Kramer test); nevertheless, a small but significant difference between hypoxic and control hearts remained (P<0.05; Tukey-Kramer test).
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Peroxide production by heart mitochondria was measured using pyruvate and malate as complex I-linked substrates and succinate as a complex II-linked substrate. The rate of peroxide production in heart mitochondria from normoxic hearts was 2-fold higher than in control rats for complex I- and II-linked substrates at the end of reperfusion (P<0.01, Fig. 6
). Hypoxic reperfusion almost completely prevented this increase (P<0.05). To better characterized the protection exerted by hypoxic reperfusion against protein oxidative damage, carbonyl groups on oxidized proteins from normoxic and hypoxic heart mitochondria were derivatized with DNPH on Oxyblot and detected using an anti-DNP antibody by Western blot, as described in Materials and Methods. Representative examples of Oxyblot are shown in Fig. 7
. A drastic decrease in oxidized proteins was detected in hypoxic rats (lines 6, 8) with normoxic rats (lines 2, 4).
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To understand the role played by mitochondrial ROS generation during reperfusion and better clarify the protection exerted by 3 min of hypoxia, we measured mitochondrial peroxide production, GSH, and GSSG 3 min after the onset of reperfusion. As shown in Fig. 5C, D
, mitochondria ROS generation under state 4 was 3-fold higher in normoxic than in the hypoxic group (P<0.001, t test). Accordingly, mitochondria GSH was largely oxidized in the normoxic group probably to scavenge ROS overproduction as demonstrated by a GSSG increase (Fig. 4C, D
). Three minutes of hypoxia completely prevented GSH oxidation and GSSG accumulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 18)
. By contrast, modifying the hydrodynamic conditions during the early period of reperfusion has been reported to decrease the extent of reperfusion injury (17
, 19
, 20)
. Therefore, mechanical manipulation of the early phase of reperfusion affords some protection against postischemic injury. Recently Zhao et al. (7)
showed that repetitive cycles of short ischemia during early reperfusion significantly reduce infarct size in a canine model. In that study, a close relationship between their findings and an oxygen-mediated process was suggested, although the source of oxidants were not identified. An increase in peroxide production after reperfusion has been described by several authors (21
22
23)
. In the present study, we have found several indices of oxidative stress elevated in mitochondria as well as in heart homogenate, such as GSSG and protein carbonyls. We found that several mechanisms contributed to this redox imbalance. It has been reported that ischemia-reperfusion enhances ROS production in heart mitochondria (24)
. However, to our knowledge this is the first study in which the rate of peroxide production has been assessed in heart mitochondria from ischemic rats under state 4 conditions using physiological substrates linked to complexes I and II. These conditions are much closer to what happens in the hearts of rats in vivo.
Our results indicate that ischemia-reperfusion causes an increase in peroxide production through mitochondrial complexes I and III, as enhancement was found with both types of substrate (i.e., pyruvate/malate and succinate). The 3-fold increase in the rate of mitochondrial peroxide production observed in the present study indicates that the contribution of mitochondria to oxidative damage in ischemia-reperfusion may have been underestimated. The high mitochondrial ROS production may explain the oxidative damage and glutathione oxidation found in the heart, particularly in mitochondria, and could be responsible for the mitochondrial impairment previously reported (11
, 25)
.
Hypoxic reperfusion prevents mitochondria impairment and reduces GSH and protein oxidation
Despite the known damaging potential of high oxygen levels, hyperoxic cardiopulmonary bypass is widely used during surgical repair of hypoxic immature and normoxic adult hearts (26)
. Earlier studies of the hypoxic immature heart suggested the existence of the damaging potential of high oxygen levels during extracorporeal circulation (27
, 28)
. Ihnken and co-workers demonstrated that oxidative myocardial damage occurring during human hyperoxic cardiopulmonary bypass can be limited by reduced oxygen tension to normoxic conditions (26
, 27)
. Our data support Ihnken’s hypothesis that further reduction of pO2 could lead to further improvement in results. In fact, our data demonstrate that a brief hypoxia applied during early reperfusion is cardioprotective by attenuating reperfusion injury. Our study agrees with the recent report by Zhao et al., who suggested that repetitive ischemia before reperfusion is as effective as preconditioning in reducing infarct size (7)
. Moreover, it was recently reported that ischemic postconditioning is able to converts persistent ventricular fibrillation in regular rhythm during human surgery (29)
and that liver postconditioning represents an alternative and effective approach to ischemia-reperfusion injury in liver surgery (12)
.
The present study documents specific involvement of mitochondria in the protection exerted by hypoxic reperfusion in heart ischemia. Recent papers from Zhao (7)
and Kin (8)
have reported that the early moments of reperfusion are important in the pathogenesis of postischemic injury and that manipulation of this early reperfusion phase could reduce the downstream consequences of ischemia reperfusion injury. Moreover, they observed in a rat model that postconditioning applied over the first minute of reperfusion optimally reduced infarct size. There is wide consensus that the protection mechanism of postconditioning involves prevention of the burst of free radical generation (30)
, but this is the first report demonstrating that hypoxic reperfusion is associated with low mitochondrial peroxide synthesis and reduced mitochondrial and tissue oxidative stress. The present study provides biochemical support to Kin et al.’s observation (8)
; in fact, we found that in normoxic conditions 3 min after the onset of reperfusion, mitochondrial generation of ROS and GSH oxidation are much higher than in controls and higher than 40 min later. Three minutes of hypoxia before classical reperfusion are able to inhibit this oxidative burst, reducing peroxide synthesis (Fig. 5)
and allowing time for scavenging "catch-up" (Fig. 4A, B
). Our data agree with previous studies reporting that a significant burst of ROS generates within the first minutes of reperfusion peaking 4–7 min after the onset of reperfusion, followed by a persistently elevated generation thereafter (31)
. It has been clearly established that oxidative stress is an early event in apoptosis and that mitochondrial oxidative stress makes the cardiomyocytes much more susceptible to ischemia-induced apoptosis (7)
. It has been reported that GSH depletion intensifies ischemia-induced cardiac dysfunction through oxidative stress (32)
. Our results show that while normoxic reperfusion after ischemia causes oxidative stress and an increase of mitochondrial peroxide production and GSH oxidation (thereby favoring apoptosis through mitochondria permeability transition), hypoxic reperfusion would protect against this cell death by preventing mitochondrial oxidative stress and increasing GSH levels.
In conclusion, in the present study we show that hypoxic reperfusion improves the mechanical recovery of rat hearts during both systole and diastole after cardioplegic ischemia and reperfusion, preventing mitochondria peroxide production, GSH oxidation, and protein damage. Hypoxic reperfusion may be clinically applicable in coronary interventions, coronary artery bypass surgery, and organ transplantation, where reperfusion injury is expressed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication May 20, 2004. Accepted for publication October 15, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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