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1Division of Experimental Cardiology, Thoraxcenter and 2Department of Biochemistry, Cardiovascular Research Institute COEUR, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
Submitted 9 February 2006 ; accepted in final form 16 November 2006
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ABSTRACT |
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0.05). In contrast, 3POC30 increased infarct size produced by CAO15 from 3 ± 1% to 19 ± 6% and CAO30 from 36 ± 6 to 48 ± 4% (both P 0.05). This deleterious effect of 3POC30 was not stimulus sensitive because postconditioning with 3POC5 and 3POC15 after CAO30 also increased infarct size. The cardioprotection by 3POC30 after CAO60 was accompanied by an increased stimulation of Akt phosphorylation at 7 min of reperfusion and a 36% lower superoxide production, measured by dihydroethidium fluorescence, after 2 h of reperfusion. Consistent with these results, cardioprotection by 3POC30 was abolished by phosphatidylinositol-3-OH-kinase inhibition, as well as nitric oxide (NO) synthase inhibition. The deleterious effect of 3POC30 after CAO15 was accompanied by an increased superoxide production with no change in Akt phosphorylation and was not affected by NO synthase inhibition. In conclusion, the effect of cardiac POC depends critically on the duration of the index ischemia and can be either beneficial or detrimental. These paradoxical effects of POC may be related to the divergent effects on Akt phosphorylation and superoxide production.
myocardial infarction; rat; reperfusion injury; reperfusion injury salvage kinase pathway; reactive oxygen species
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METHODS |
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Experiments were performed in male Wistar rats (300–380 g) in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and with prior approval of the Animal Care Committee of the Erasmus MC, University Medical Center Rotterdam.
Surgical and Experimental Procedures
Pentobarbital sodium-anesthetized (60 mg/kg ip) rats were intubated for positive pressure ventilation with oxygen-enriched room air. Through the carotid artery, a polyethylene (PE)-50 catheter was positioned in the thoracic aorta for measurement of arterial blood pressure and heart rate (10, 21–23, 33). In the inferior caval vein, a PE-50 catheter was placed for infusion of Gelofusin (5–10 ml; B. Braun Melsungen), to maintain central venous pressure at 4–6 mmHg, and for administration of drugs. After a thoracotomy was performed via the left third intercostal space, the pericardium was opened and a silk 6-0 suture was looped under the left anterior descending coronary artery for later occlusion of the vessel. A catheter was positioned in the abdominal cavity to allow intraperitoneal administration of pentobarbital for maintenance of anesthesia. Rectal temperature was maintained at 36.5–37.5°C (10, 22, 33).
Rats that fibrillated during occlusion or reperfusion were allowed to complete the protocol, provided that conversion to normal sinus rhythm occurred spontaneously within 1 min or that defibrillation via gently thumping on the thorax was successful within 2 min after onset of fibrillation. Occlusion and reperfusion were visually verified. The area at risk (AR) and infarct area (IA) were determined after 120 min of reperfusion, using Trypan blue and Nitro-Blue-Tetrazolium staining, respectively (10, 22, 33). Infarct size (IS) was calculated as IA/AR x 100.
Experimental Protocols/Design
After completion of surgery, a 30-min stabilization period was allowed before animals were subjected to the experimental protocols (see Fig. 1 for overview of protocols). The duration of this stabilization period has been shown to be sufficiently long to exclude an effect of the surgical procedures on the development of infarct size (28).
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Protocol 2: importance of the POC stimulus. In another set of experiments, we investigated the influence of the POC stimulus. For this purpose, CAO30 and CAO60 were followed by a postconditioning stimulus consisting of three cycles of 5 s (3POC5) or three cycles of 15 s (3POC15) of reperfusion and reocclusion.
Protocol 3: involvement of PI3K-Akt-eNOS signaling pathway in POC. Four additional groups of rats were subjected to either 15 or 60 min of index ischemia followed by either abrupt reperfusion or 3POC30. After 7 min of abrupt reperfusion (control) or after postconditioning with 3POC30 followed by 7 min of reperfusion (3POC30) (7, 32), the AR was dissected and snap frozen in liquid nitrogen within 30 s before being stored at –80°C. The 7 min reperfusion time point was employed on the basis of previous POC studies (2, 4, 14, 15, 32) and in view of a previous in vitro study, in which Akt phosphorylation increased four- to fivefold within 10 min of reperfusion and remained elevated up to 60 min (24). Furthermore, phosphorylation (P) of both (Ser473)-Akt and (Thr308)-Akt has recently been shown to be obligatory for the full activation of Akt (36). Consequently, we determined P(Thr308)-Akt as well as P(Ser473)-Akt.
Additional rats were subjected to CAO60 with or without 3POC30 in the presence of the PI3K inhibitor wortmannin (15 µg/kg iv) (11) or the NO synthase inhibitor N
-nitro-L-arginine (L-NNA; 25 mg/kg iv) (23).
Protocol 4: involvement of ROS in POC. Four separate groups of animals were subjected to CAO15 or CAO60 followed by either abrupt reperfusion or 3POC30, while one group of control animals underwent only a sham procedure, i.e., without index ischemia. The four groups subjected to the 15-min or 60-min index ischemia were studied at the end of 120 min of reperfusion (39).
Two additional groups of rats underwent CAO15 either without or with 3POC30 (followed by 2 h of reperfusion), including treatment with the combination of ROS scavengers N(-2-mercaptopropionyl)-glycine (MPG; 30 mg·kg–1·h–1 iv), N-acetylcysteine (NAC; 250 mg·kg–1·h–1 iv), and Tempol (50 mg·kg–1·h–1 iv). Infusion was started 15 min before the onset of the CAO15 and was maintained throughout the experimental protocol. Starting 5 min before the onset of initial reperfusion, the infusion rates were doubled for 15 min.
Biochemical Assays
Akt phosphorylation. Akt phosphorylation was determined in the AR harvested at 7 min of reperfusion. Approximately 40 mg of frozen tissue were homogenized at liquid nitrogen temperature in a microdismembrator unit (B. Braun Biotech International). The frozen powder was suspended in 20 volumes of cold Laemmli loading buffer (20), and, thereafter, the suspensions were heated for 5 min at 95°C and sonicated in a Bioruptor. Protein determination was done using the RCDC protein assay (Bio-Rad). Proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Immunblot, Bio-Rad). Blots were preincubated in Tween-20 Tris-base sodium chloride [10 mmol/l Tris·HCl (pH 7.6), 150 mmol/l NaCl, and 0.1% Tween-20] supplemented with 5% BSA and incubated with diluted primary antibodies against Akt or phospho(Thr308)-Akt (rabbit polyclonal, Cell Signaling) or phospho(Ser473)-Akt (mouse monoclonal, New England Biolabs). Blots were probed with horseradish peroxidase-conjugated goat or mouse anti-rabbit secondary antibody. Signals were visualized using Supersignal West Femto Maximum Sensitivity Substrate (Pierce) and Hyperfilm enhanced chemiluminescence (ECL, Amersham Biosciences). Signal densities were quantified using a Bio-Rad calibrated GS-800 scanner.
Dihydroethidium fluorescence. Superoxide anion generation from ischemic-reperfused myocardium was determined by using dihydroethidium (DHE) fluorescence (27, 39). Hearts were excised and left ventricular transmural tissue samples were placed in ice-cold saline, embedded in Tissue-Tek (while marking the AR), frozen in liquid nitrogen cooled isopentane, and stored at –60°C. Tissue sections of 5 µm were cut by using a cryostat, thaw mounted on Fisher-Plus (Fisher Scientific) slides, and stained with 10 µM DHE at 37°C for 30 min. Fluorescent images were obtained with a 585-nm long-pass filter. Generation of superoxide by tissue was demonstrated by red fluorescent labeling. Images were analyzed on a microscopy image analysis system (Impak C, Clemex vision image analysis system, Clemex Technologies, Longueuil, QC, Canada) on which a subroutine has been written to assess the total fluorescence per slide to quantify the amount of radical damage. At least five determinations were performed in each group.
Data Analysis and Presentation
Infarct size data were analyzed using two-way (duration of index ischemia x POC) ANOVA followed by post hoc testing with Student-Newman-Keuls test. Heart rate and arterial blood pressure were analyzed using two-way (time x treatment) ANOVA for repeated measures followed by Student-Newman-Keuls test. SDS-PAGE allows loading of only 12 samples per gel. Therefore, the signal intensities on the immunoblot, expressed as fold increase compared with corresponding controls on the same gel, were analyzed using Student-Newman-Keuls test. Statistical significance was accepted when P 0.05. All data are presented as means ± SE.
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RESULTS |
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Of the 271 rats that entered the study, nine rats were excluded due to technical failure, five rats were excluded due to an AR <10% of the left ventricle, and four rats died prematurely during the index ischemia because of pump failure (no more than 1 rat per group).
Importance of the Duration of Index Ischemia
Infarct size. There were no intergroup differences in the AR (40 ± 1%; P = 0.56) between the experimental groups (data not shown). Increasing the duration of index ischemia from 15 to 120 min produced a progressively greater infarct size in the animals of the control group, reaching a plateau after CAO60 (Fig. 2). Postconditioning with 3POC30 was cardioprotective when the stimulus was applied after CAO45 and CAO60, but it was ineffective when applied after coronary occlusions of longer duration (CAO90 and CAO120). Conversely, when the 3POC30 stimulus was applied following CAO15 and CAO30, infarct size was increased compared with their respective control groups (Fig. 2).
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The additional experiments using a postconditioning stimulus with shorter reocclusion-reperfusion cycles revealed that the increase in infarct size by 3POC30 following CAO30 was not specific for this particular postconditioning stimulus, because infarct size was similarly increased when CAO30 was followed by 3POC5 (P = 0.15 vs. control) or 3POC15 (P < 0.05 vs. control; Fig. 3). Furthermore, there was no significant difference between the effects of the three different POC stimuli (P > 0.20). However, there was a significant difference compared with control when all three POC stimuli were combined compared with control (P < 0.01). Conversely, both 3POC5 (P = 0.13) and 3POC15 (P = 0.18) failed to emulate the protection against CAO60 that was observed with 3POC30 (P < 0.05). Also, in the CAO60 groups, there was no significant difference between the effects of the three different POC stimuli (P > 0.20), whereas there was a significant difference compared with control when all three POC stimuli were combined (P < 0.05).
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Akt phosphorylation. Total Akt was unchanged at 7 min of reperfusion after CAO15 as well as CAO60. However, there were marked increments in P(Thr308)-Akt (10- and 7-fold, respectively) and P(Ser473)-Akt (8- and 4-fold, respectively) (Fig. 4, A and B). Postconditioning with 3POC30 following CAO60 produced further increase in P(Thr308)-Akt, but the further increase in P(Ser473)-Akt failed to reach statistical significance (Fig. 4, A and C); these increases were attenuated in the presence of the PI3K-inhibitor wortmannin (example shown in Fig. 4A). Conversely, following CAO15, 3POC30 did not increase but rather tended to decrease levels of P(Thr308)-Akt and P(Ser473)-Akt (Fig. 4C).
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0.05). Involvement of ROS in POC
DHE fluorescence. DHE reacts with superoxide anions to form ethidium bromide, which, in turn, intercalates with DNA to provide nuclear fluorescence as a marker for superoxide anion generation. As shown in Fig. 6, DHE fluorescence was markedly enhanced at 120 min of reperfusion following CAO60 compared with sham-operated animals (sham); 3POC30 attenuated the DHE fluorescence at 120 min of reperfusion following CAO60 by 36% (Fig. 6, C and D). DHE fluorescence was not affected at 120 min of reperfusion by CAO15 compared with sham, which is consistent with observations that the burst of ROS occurs principally during the first few minutes of reperfusion following CAO15 (3). Conversely, 3POC30 following CAO15 increased DHE fluorescence at 120 min of reperfusion by 12%, compared with CAO15 alone.
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DISCUSSION |
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Importance of Index Ischemia Duration
Currently, it is incompletely understood what the optimal duration of the reocclusion and reperfusion periods of POC is and to what extent its cardioprotective effect depends on the duration of index ischemia, experimental conditions or animal species (34). For example, while four 30-s periods of abrupt CAO starting 30 s after the initial reperfusion, and interspersed by 30 s of abrupt reperfusion, limited infarct size produced by a 30-min CAO in the in situ rabbit heart (38), it failed to afford significant protection in the isolated buffer-perfused rabbit heart (37). Furthermore, while three 30-s periods of abrupt CAO starting 30 s after the initial reperfusion, and interspersed by 30 s of abrupt reperfusion, limited infarct size produced by a 60-min CAO in the dog (13, 39), it failed to afford cardioprotection against a 30-min CAO in the rat (34). The latter authors proposed that, in small rodents, briefer periods (i.e., 10–15 s) of reocclusion and reperfusion are required, whereas in larger animals, 30-s cycles are effective (34). In discordance with this notion, however, a recent study in swine showed that three cycles of 30 s of reocclusion and reperfusion failed to afford protection against infarct size produced by a 30-min CAO (29). Since the 30-s algorithm was effective against a 60-min CAO (13, 39), but not always against a 30-min CAO (29, 34, 37), we hypothesized that another important determinant of the optimal algorithm could be the duration of index ischemia.
The present study demonstrates that the effect of POC on myocardial infarct size in the rat heart depends critically on the duration of the index ischemia. Thus, while cardioprotection was observed with 3POC30 following the 45- and 60-min CAO, protection was lost with the longer occlusion durations of 90 and 120 min. This loss of protection likely reflects the progressive contribution of ischemic damage to infarction, with little contribution of reperfusion injury, when the duration of index ischemia is prolonged. Paradoxically, we observed that with 15-min CAO (which elicited negligible infarction under control conditions) as well as with 30-min CAO, 3POC30 aggravated irreversible damage. The increase in infarct size after postconditioning with 3POC30 was not a consequence of the application of this stimulus in a small animal model because postconditioning with 3POC5 or 3POC15, purportedly appropriate stimuli for small animals (34), also increased infarct size. Nevertheless, our data differ from that study (34), in which it was shown that postconditioning with either 3POC10 or 3POC15 after 30-min CAO limited infarct size, while postconditioning with 3POC30 had no effect (34). An explanation for these different observations is not readily found, but there are some differences between the two studies. First, although both studies employed male rats, we used Wistar rats, whereas Vinten-Johansen et al. (34) used Sprague-Dawley rats. Another difference in experimental design is the use of isoflurane in addition to the pentobarbital anesthesia in the study by Vinten-Johansen et al. (34). This could be important, because it has been reported that isoflurane can protect the myocardium against irreversible reperfusion damage at least, in part, by activation of PI3K-signaling (6, 8, 35). Nevertheless, our study indicates that the effect of POC not only depends on the algorithm of POC but also on the duration of index ischemia. The present data may also help to understand why, in pigs, postconditioning CAO30 with 3POC30 was not cardioprotective (29), while myocardial postconditioning in this species with the same stimulus after CAO75 was effective (17). Furthermore, it may help to explain why, in rats, in one study, 6POC10 afforded protection against CAO30 (34) but failed to protect against CAO60 (31).
Involvement of PI3K-Akt-eNOS Signaling Pathway and ROS
The novel finding that the effect of POC on infarct size could be double edged, i.e., beneficial or detrimental, depending on the duration of the index ischema, warranted further investigation on the molecular mechanisms involved. There is already substantial evidence that several reperfusion injury survival kinase (RISK) pathways play a role in the cardioprotection by POC (34). For example, studies in the rabbit indicate that POC activates ERK1/2 (7), while the protection by POC is abolished by the ERK1/2 inhibitor PD-98059 (7, 38). On the other hand, the PI3K-Akt prosurvival pathway has been implicated in the protection in the isolated buffer-perfused rabbit (37) and rat (32) heart. The present study extends those observations to the in vivo rat heart and shows that POC following CAO60 not only further increases Akt-phosphorylation, but also that the reduction of infarct size is abolished by the selective PI3K inhibitor wortmannin (11). Further evidence for the involvement of the PI3K-Akt prosurvival pathway is the abolition of the POC-induced protection by the NO synthase inhibitor L-NNA, which corroborates previous findings in the in vivo rabbit heart (38). How the PI3K-Akt-eNOS pathway exerts its protection cannot be derived from the present observations, but there is evidence that preventing the opening of the mitochondrial permeability transition pore (mPTP) forms a crucial step in mediating the protection by POC (2, 4, 14, 15, 32). NO can inhibit opening of the mPTP directly but also indirectly by scavenging superoxide. In agreement with previous observations (19, 39), it was observed in the present study that 3POC30 attenuated the superoxide anion production at 120 min of reperfusion, which was likely due to an increased NO production by activation of the PI3K-Akt-eNOS pathway.
Paradoxically, 3POC30 following CAO15 increased infarct size. This was at variance with 3POC30 following CAO60, accompanied by an unchanged activity of the PI3K-Akt prosurvival pathway. Inhibition of NO synthase had no effect on the aggravation of irreversible injury produced by 3POC30, which is consistent with the lack of further increase of stimulation of the PI3K-Akt-eNOS prosurvival pathway. The apparent lack of NOS activation may also explain why we observed an increase, rather than a decrease, in superoxide anion production at 120 min of reperfusion when POC followed CAO15. Administration of a combination of ROS scavengers abolished the damage by 3POC30 after CAO15, indicating that the increased oxidative stress produced by the intermittent reocclusion and reperfusion sequences, following the shorter periods (15 and 30 min) of index ischemia, contributes to the increase in infarct size. Indeed, in the case of the 15-min index ischemia, only 3% infarct size is observed under control conditions, which increased to 19% when 3POC30 was applied. The mechanism by which oxidative stress aggravates necrosis under this condition cannot be derived from the present study and should be the subject of future investigations.
Conclusions
The effect of POC on myocardial infarct size is ambiguous. Although most studies, including one in humans (30), have shown that POC is cardioprotective (2, 7, 18, 19, 32, 38), there are several reports that have failed to observe a cardioprotective effect (29, 34, 37). This discrepancy has been ascribed to the employed POC algorithm in relation to the species studied. The present study demonstrates that the cardiac effects of POC may even be detrimental and that this deleterious effect depended critically on the duration of the preceding period of index ischemia rather than the employed stimulus. These paradoxical effects of POC are best explained by the distinct effects on Akt phosphorylation and superoxide anion production.
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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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