HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 288/4/H1900    most recent 01244.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (47) Citing Articles via Google Scholar Google Scholar Articles by Sun, H.-Y. Articles by Zhao, Z.-Q. Search for Related Content PubMed PubMed Citation Articles by Sun, H.-Y. Articles by Zhao, Z.-Q.

Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca²⁺ overload

Cardiothoracic Research Laboratory, Carlyle Fraser Heart Center, Crawford Long Hospital, Emory University School of Medicine, Atlanta, Georgia

Submitted 31 December 2003 ; accepted in final form 21 November 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
We have shown that intermittent interruption of immediate reflow at reperfusion (i.e., postconditioning) reduces infarct size in in vivo models after ischemia. Cardioprotection of postconditioning has been associated with attenuation of neutrophil-related events. However, it is unknown whether postconditioning before reoxygenation after hypoxia in cultured cardiomyocytes in the absence of neutrophils confers protection. This study tested the hypothesis that prevention of cardiomyocyte damage by hypoxic postconditioning (Postcon) is associated with a reduction in the generation of reactive oxygen species (ROS) and intracellular Ca²⁺ overload. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h of hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned after the 3-h index hypoxia by three cycles of 5 min of reoxygenation and 5 min of rehypoxia applied before 6 h of reoxygenation. Relative to sham control and hypoxia alone, the generation of ROS (increased lucigenin-enhanced chemiluminescence, SOD-inhibitable cytochrome c reduction, and generation of hydrogen peroxide) was significantly augmented after immediate reoxygenation as was the production of malondialdehyde, a product of lipid peroxidation. Concomitant with these changes, intracellular and mitochondrial Ca²⁺ concentrations, which were detected by fluorescent fluo-4 AM and X-rhod-1 AM staining, respectively, were elevated. Cell viability assessed by propidium iodide staining was decreased consistent with increased levels of lactate dehydrogenase after reoxygenation. Postcon treatment at the onset of reoxygenation reduced ROS generation and malondialdehyde concentration in media and attenuated cardiomyocyte death assessed by propidium iodide and lactate dehydrogenase. Postcon treatment was associated with a decrease in intracellular and mitochondrial Ca²⁺ concentrations. These data suggest that Postcon treatment reduces reoxygenation-induced injury in cardiomyocytes and is potentially mediated by attenuation of ROS generation, lipid peroxidation, and intracellular and mitochondrial Ca²⁺ overload.

reactive oxygen species; hypoxia; reoxygenation; superoxide; ischemia; reperfusion

Our laboratory recently demonstrated that postconditioning, defined as a repetitive series of brief interruptions of reperfusion applied at the immediate onset of reperfusion, significantly reduced myocardial injury in canine (18, 53) and rat (29) models of reversible coronary occlusion. This cardioprotection was comparable but not additive to ischemic preconditioning (18). Therefore, postconditioning is a rapidly evoked, endogenous, protective mechanism that exerts protection during the very early minutes of reperfusion. It has been suggested from in vivo studies that the cardioprotective effects of postconditioning are derived from inhibition of the neutrophil-mediated inflammatory response during early reperfusion (18, 52). However, some studies suggest that cardioprotection exerted by postconditioning is partially independent of inflammatory cells, because it can be elicited in isolated buffer-perfused heart preparations (40, 50). The ability of postconditioning to attenuate ROS generation by cardiomyocytes independent of neutrophils and other inflammatory cells has not been investigated. Furthermore, it has not been determined whether the cardioprotection of postconditioning involves a reduction in myocyte Ca²⁺ accumulation. Finally, it has not been determined whether a postconditioning-like phenomenon can be elicited after hypoxia and reoxygenation. Accordingly, we hypothesized that hypoxia-reoxygenation-induced cardiomyocyte damage is attenuated in association with limiting ROS generation and intracellular Ca²⁺ overload when cultured cardiomyocytes are postconditioned during the early phase of reoxygenation with repetitive cycles of reoxygenation and hypoxia, i.e., hypoxic postconditioning (Postcon) in the absence of inflammatory cells and subsequent endothelial cell-cell interaction-mediated events.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The experimental animals were handled in compliance 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). The study protocol was approved by the Institutional Animal Care and Use Committee of Emory University.

Isolation of neonatal rat cardiomyocytes. Primary cultures of neonatal rat cardiomyocytes were prepared from 1- to 3-day-old Wistar rats. The hearts were rapidly excised, minced, and dissociated with 0.08% trypsin. The dispersed cells were then plated at a field density of 2 x 10⁵ cells/cm² on 60-mm culture dishes with DMEM supplemented with 10% fetal bovine serum (FBS) for a total volume of 5 ml. After 24 h of plating in an incubator with 95% O₂-5% CO₂ at 37°C, the culture medium was changed to DMEM with 10% FBS that contained 10 µM cytosine arabinoside to eliminate noncardiomyocytes. After hypoxia, the culture medium was replaced with fresh oxygenated DMEM, and the dishes were transferred to a normoxic incubator (95% air-5% CO₂) for 6 h of reoxygenation (45).

Experimental protocols. After 5–6 days of cell culture in normoxic DMEM, the culture medium was freshly changed with 1% FBS-DMEM, and the cardiomyocytes were randomly divided into three groups as follows: 1) sham control (sham), whereby cardiomyocytes were seeded on the plate for a total of 9 h of normoxic incubation; 2) hypoxia-reoxygenation, in which the culture dishes were transferred to a hypoxic incubator in a humidified atmosphere that contained 95% N₂-5% CO₂ for 3 h of hypoxia (PO₂ range in the incubator was maintained at <1%), and, subsequently, 6 h of reoxygenation; and 3) Postcon, in which postconditioning of cardiomyocytes was achieved using two different incubators (hypoxic and normoxic); at the end of 3 h of hypoxia, the cardiomyocytes were initially transferred to a normoxic incubator for 5 min and then returned to the hypoxic incubator for an additional 5 min without a change in culture medium. The postconditioning cycle was repeated three times and followed by 6 h of continuous normoxia (i.e., reoxygenation). In the Postcon group, each cycle of postconditioning hypoxia reduced the PO₂ of the medium by 60% relative to that seen at the end of each cycle of reoxygenation (Fig. 1). In contrast, PO₂ in the culture medium of the hypoxia-reoxygenation group was restored to the baseline level over 20 min.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Partial pressure of oxygen (PO2) in the culture medium after 3 h of hypoxia (H) followed by 30 min of reoxygenation (Re) during postconditioning (Post-con) period. PO2 of the medium was reduced during hypoxia and returned to baseline values at 20 min of reoxygenation. Each cycle of Post-con treatment decreased (during hypoxia) and increased (during reoxygenation) media PO2.

Lactate dehydrogenase release. The amount of lactate dehydrogenase (LDH) released into the culture medium from the injured cells after 6 h of reoxygenation was assayed using a Sigma assay kit. The optical density of the tetrazolium product was determined spectrophotometrically (Molecular Devices; Sunnyvale, CA) at a 490-nm wavelength. The maximum LDH release was expressed as the percent activity from sham cells.

Superoxide anion production by lucigenin-enhanced chemiluminescence. Lucigenin, a compound that emits light upon interaction with superoxide anion, was used to quantify superoxide anion production from cardiomyocytes as described previously (37). Specifically, the cardiomyocytes (1 x 10⁶ cells for each assay) harvested from different groups at the end of the 30-min reoxygenation were suspended in a polypropylene tube that contained 0.5 ml of phosphate-buffered saline and 5 µM lucigenin; this concentration avoids recycling reactions. The lucigenin-enhanced chemiluminescence reactions were read over 3 min in a luminometer (Autolumat Plus LB953; Berthold Tech). Photomultiplier background signals were automatically subtracted. Superoxide anion generation values were expressed as relative light units (RLUs).

Quantitation of superoxide anion release by cytochrome c reduction. Production of superoxide anion was also measured as the SOD-inhibitable reduction of cytochrome c (54). Briefly, after 50 µl of cytochrome c (40 µM) was mixed with 200 µl of culture media samples, 250 µl HBSS (5 mM Ca²⁺ and 1.5 mM Mg²⁺) was immediately added to the mixture. In control samples, 50 µl SOD (100 µg/ml) was added. The reaction was allowed to proceed for 10 min at room temperature after which absorbance due to cytochrome c reduction was measured spectrophotometrically at a 550-nm wavelength. Reduction of cytochrome c was calculated using a molar absorption coefficient of 21,000 mol/cm (54). The results were expressed as micromoles per gram of protein.

Quantitation of hydrogen peroxide. Hydrogen peroxide in the medium was determined using a commercial Amplex red hydrogen peroxide assay kit (Molecular Probes). In the presence of peroxidase, Amplex red reagent reacts with hydrogen peroxide in a 1:1 stoichiometry to produce the fluorescent-red oxidation product resorufin. Briefly, the cardiomyocytes harvested after the experiment were initially lysed by three cycles of freeze-thawing, which was followed by centrifugation at 3,000 g for 10 min at 4°C. The supernatant was reacted with working solution (100 µM Amplex red reagent and 0.2 U/ml horseradish peroxidase in a 1x reaction buffer) in 96-well plates and was incubated at room temperature for 30 min. Absorbance of the color reaction was determined spectrophotometrically (Molecular Devices) at a 560-nm wavelength. The results were expressed as micromoles per gram of protein.

Determination of lipid peroxidation. Lipid peroxidation was estimated by measuring the concentration of malondialdehyde (MDA) in the isolated myocytes using a Lipid Peroxidation Assay Kit (Calbiochem; La Jolla, CA). Briefly, the cells were lysed by repetitive freeze-thawing and were mixed with reagent R1 (N-methyl-2-phenylindole in acetonitrile and methanol) for a total of 60 min at 45°C. The samples were centrifuged at 15,000 g for 10 min to clarify the supernatant just before being read, and the absorbance of the supernatant was determined by a spectrophotometer (Molecular Devices) at a 586-nm wavelength. The MDA concentration was calculated using standard curves and values were expressed as micromoles per gram of protein.

Measurement of intracellular and mitochondrial Ca²⁺ concentrations. Intracellular and mitochondrial Ca²⁺ concentrations ([Ca²⁺]_i and [Ca²⁺]_m, respectively) were determined as previously described (36, 45) and according to the manufacturer's instructions (Molecular Probes). In brief, after the experiment, the cardiomyocytes (1 x 10⁶ cells/sample) that were cultured in the 12-well plate or slides were initially washed with HEPES buffer that contained (in mM) 130 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 10 HEPES, 11 glucose, and 0.2 CaCl₂ at pH 7.4 and were then stained using 5 µM fluo-4 AM for [Ca²⁺]_i or 5 µM X-rhod-1 AM for [Ca²⁺]_m for 30 min at room temperature. To avoid deesterification of intracellular X-rhod-1 AM in the cytosolic compartment, which would interfere with detection of [Ca²⁺]_m (36), the cardiomyocytes were rinsed and incubated with 100 µM MnCl₂-HEPES for an additional 20 min to quench the cytosolic Ca²⁺ signal.

Fluorescence measurement of Ca²⁺ was determined using a fluorescence plate reader (CytoFluor II; PerSeptive Biosystems; Framingham, MA) at excitation wavelengths of 485 nm for [Ca²⁺]_i and 580 nm for [Ca²⁺]_m and emission wavelengths of 530 nm for [Ca²⁺]_i and 645 nm for [Ca²⁺]_m using the following equation: [Ca²⁺]_i = K_d[(F – F_min)/(F_max – F)], where K_d is the dissociation constant (345 nM for fluo-4 and 700 nM for X-rhod-1), F is the fluorescence at intermediate Ca²⁺ levels (corrected from background fluorescence), F_min is the fluorescence intensity of the indicator in the absence of Ca²⁺ and is obtained by adding a solution of 10 mM EGTA for 15 min, and F_max is the fluorescence of the Ca²⁺-saturated indicator and is obtained by adding a solution of 25 µM of digitonin in 2.2 nM CaCl₂ for 15 min (45). Final values for [Ca²⁺]_i and [Ca²⁺]_m were expressed in nanomoles.

To validate the measurement of [Ca²⁺]_m, the cultured cardiomyocytes were transferred into a slide chamber after X-rhod-1 AM staining and were placed on the stage of a fluorescence microscope (x50 objective; Olympus). The images from the slides were captured using a digital camera connected to and analyzed by Image-Pro Plus software (Media Cybernetics; Silver Spring, MD). Fluorescence intensity of [Ca²⁺]_m (subtracted from background fluorescence) was determined, and the fluorescence intensities of the images were expressed as arbitrary units per millimeter-square field as previously described (41).

Statistical analysis. All experiments were repeated at least four separate times. Within each experiment, either duplicate or triplicate plates were analyzed for each parameter observed. Single time-point variables were analyzed by ANOVA followed by Student-Newman-Keuls test for multiple comparisons. Time-dependent comparisons among groups were analyzed by repeated-measures ANOVA followed by post hoc analysis with Student-Newman-Keuls for multiple comparisons. A P value <0.05 was considered significant. All values are expressed as means ± SE.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cardiomyocyte viability and LDH release. Hypoxia-reoxygenation significantly increased the number of dead cardiomyocytes as evidenced by an elevated peak and area under the curve of sub-G1 fraction compared with the sham control group (Fig. 2). When cardiomyocytes were postconditioned at the onset of reoxygenation by brief, repeated cycles of reoxygenation-hypoxia, the number of dead cardiomyocytes was reduced on average by 45% compared with the hypoxia-reoxygenation group. LDH leakage (percentage of sham control) in the culture medium determined after hypoxia-reoxygenation was significantly higher than in the sham control group (155 ± 9 vs. 99 ± 6 in sham control; P < 0.01; Fig. 3). LDH leakage in the medium was significantly reduced when cardiomyocytes were postconditioned before full reoxygenation (101 ± 6; P < 0.01 vs. hypoxia-reoxygenation group). The reduction in cell death assessed by LDH activity in the medium is consistent with the reduction in the number of PI-positive cardiomyocytes.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Representative flow cytometric analysis of primary neonatal rat cardiomyocyte death in the sham control (top left), hypoxia-reoxygenation (H/Re; top right), and postconditioning (bottom left) groups. Percentage of total cell death was determined by propidium iodide (PI) staining (bottom right). Treatment group conditions were as follows: sham group, 9 h of cell culture in normoxic conditions; hypoxia-reoxygenation group, 3 h of hypoxia and 6 h of reoxygenation; and Post-con group, three cycles of 5-min reoxygenation and 5-min hypoxia after the 3-h index hypoxia but before 6 h of reoxygenation. Peak of sub-G1 fraction is an indicator of the number of dead cells. FL2-H, fluorescent intensity of propidium iodide. Each value represents the group mean ± SE of at least six independent experiments. *P < 0.05 vs. sham control group; P < 0.05 vs. hypoxia-reoxygenation group.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Lactate dehydrogenase (LDH) activity in sham control, hypoxia-reoxygenation, and Post-con groups. Post-con treatment significantly reduced LDH activity relative to hypoxia-reoxygenation group. Bars represent group means ± SE of at least eight independent experiments. *P < 0.05 vs. sham control group; P < 0.05 vs. hypoxia-reoxygenation group.

View larger version (22K): [in this window] [in a new window]   Fig. 4. A: kinetics of lucigenin-enhanced chemiluminescence (CL) determined over 3 min from the cultured cardiomyocytes (1 x 106 cells) after hypoxia and reoxygenation. B: peak in elevation of lucigenin-enhanced chemiluminescence was significantly reduced when the cardiomyocytes were postconditioned (Post-con group) during early reoxygenation relative to hypoxia-reoxygenation group. Values are expressed as relative light units (RLUs). *P< 0.05 vs. sham control group; P < 0.05 vs. hypoxia-reoxygenation group.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Superoxide anion production measured as the SOD-inhibitable reduction of cytochrome c after hypoxia and reoxygenation. Post-con treatment significantly attenuated cytochrome c reduction relative to hypoxia-reoxygenation group. Bars represent group means ± SE of at least six independent experiments. *P < 0.01 vs. sham control group; P < 0.05 vs. hypoxia-reoxygenation group.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Hydrogen peroxide (H2O2) concentration (A) and malondialdehyde (MDA) levels (B) in cardiomyocytes after 3 h of hypoxia and 6 h of reoxygenation. Post-con treatment significantly attenuated hydrogen peroxide generation and reduced malondialdehyde levels relative to hypoxia-reoxygenation group. Bars represent group means ± SE of at least 10 independent experiments. *P < 0.05 vs. sham control group; P < 0.05 vs. hypoxia-reoxygenation group.

View larger version (17K): [in this window] [in a new window]   Fig. 7. A: intracellular Ca2+ concentration ([Ca2+]i) was detected by fluorescent fluo-4 AM staining. After 3 h of hypoxia and 1 h of reoxygenation, [Ca2+]i was increased relative to the sham control group. Post-con treatment at reoxygenation significantly reduced [Ca2+]i relative to the hypoxia-reoxygenation group. Bars represent group means ± SE of at least eight independent experiments. *P < 0.01 vs. sham control group; P < 0.05 vs. hypoxia-reoxygenation group. B: mitochondrial Ca2+ concentration ([Ca2+]m) was measured by fluorescent X-rhod-1 AM staining. After 3 h of hypoxia and 6 h of reoxygenation, [Ca2+]m was significantly increased relative to the sham control group. Post-con treatment significantly reduced [Ca2+]m relative to the hypoxia-reoxygenation group. *P < 0.01 vs. sham control group; P < 0.05 vs. hypoxia-reoxygenation group.

View larger version (70K): [in this window] [in a new window]   Fig. 8. A typical fluorescence microscopic image shows superoxide anion production from cardiomyocytes after 3 h of hypoxia and 1 h of reoxygenation. Binding of ethidium from dihydroethidium produced red fluorescence, which represents superoxide production. Ethidium nuclear staining was considerably less intense in postconditioned cardiomyocytes. This image is representative of at least four experiments (magnification, x400). Arb.u, arbitrary units. *P < 0.05 vs. sham control group; P < 0.05 vs. hypoxia-reoxygenation group; n = 10 fields.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

The generation of ROS is a major mechanism of cell injury after ischemia-reperfusion. The failure of oxygen radical scavengers to consistently prevent the physiological effects of ROS-mediated injury has to some extent challenged the importance of ROS in ischemia-reperfusion injury. Although ROS can be detected during both ischemia and hypoxia alone (10), a robust burst of ROS has been reported after reoxygenation (27, 55) or in vivo reperfusion (9). In the in vivo situation, ROS are derived in large part from inflammatory cells (i.e., neutrophils) and neutrophil-endothelial interactions (2, 9). However, inflammatory cells are absent from isolated and cultured cell systems, and therefore ROS are generated by cardiomyocytes (31). In the present study, a significant increase in ROS was observed during reoxygenation of isolated cardiomyocytes in the absence of inflammatory cells. Postconditioning reduced this production of ROS and lipid peroxidation products.

The observation from this study that a reduction in cell death was associated with less ROS generation and less Ca²⁺ accumulation (both cytosolic and mitochondrial) may suggest a causative link between these events. Many previous reports (1, 22, 35) have shown that both generation of ROS and intracellular Ca²⁺ overload play crucial roles in the induction of cell death during reperfusion and reoxygenation. A burst of ROS increases the Ca²⁺ influx through L-type Ca²⁺ channels and leads to cytosolic Ca²⁺ accumulation (3, 11, 12, 33). In addition, ROS have been reported to increase activity of the Na⁺/H⁺ exchanger (42) and thereby lead to an increase in cytosolic Ca²⁺ via reversal of the Na⁺/Ca²⁺ antiporter. Furthermore, Ca²⁺ accumulation may occur secondary to decreased Ca²⁺ sequestration via sarcoplasmic reticular Ca²⁺- ATPase (23). Hence the accumulation of Ca²⁺ in both the cytosolic and mitochondrial compartments by multiple pathways has been linked to the pathogenesis of necrosis (38).

Increases in cytosolic Ca²⁺ and ROS generation have both been shown to be primary stimuli for opening the mPTP (16, 20). Indeed, opening of the mPTP may be a pivotal event in the transition from reversible to irreversible injury during reperfusion (8, 15, 26, 32, 49). The increase in ROS generation and mitochondrial Ca²⁺ accumulation during reperfusion or reoxygenation is consistent with the timing of the mPTP opening during the early moments of reperfusion (19). Although other events during reperfusion can promote opening of the mPTP such as rapid realkalinization, the abrupt rise in cytosolic Ca²⁺ has been shown to be sufficient to promote opening of the pore (16, 17). Accordingly, a reduction of either ROS generation or intracellular Ca²⁺ may be involved in the attenuation of cell death. By inference, this may have been a mechanism of protection with postconditioning. However, it was not determined whether Ca²⁺ overload or increased levels of ROS was the triggering event during reoxygenation of isolated cardiomyocytes. Evidence supports the idea that the generation of ROS occurs first and then induces intracellular Ca²⁺ overload (3, 11, 30, 33).

In the present study, postconditioning reduced the generation of ROS (superoxide anions and hydrogen peroxide); this was also associated with a decrease in lipid peroxidation products (MDA), which is a presumptive measure of ROS-mediated injury as well as membrane leakage of LDH. It was not determined whether ROS generation during the early moments of reoxygenation was reduced during the postconditioning period as supported by the lucigenin-enhanced chemiluminescence data during a later phase of the reoxygenation. Ostensibly, the early generation of ROS could be reduced during the postconditioning procedure by limiting the actual supply of substrate oxygen (substrate limitation) or by attenuating the enzymes involved in oxidant generation such as xanthine oxidase or endothelial NAD(P)H oxidase. A limitation of substrate oxygen as a mechanism of attenuating the ROS generation by postconditioning would be consistent with studies that report a reduction of postischemic or posthypoxic injury by low levels of oxygen perfusates (4, 5, 24, 34). The level of hypoxia achieved during the 3-h index hypoxia was sufficient to elicit cellular damage, which was expressed as a number of hallmark pathological markers. However, whether the level of hypoxia achieved during the postconditioning interval (40 mmHg) was sufficient to limit oxygen diverted from basal and recovery metabolism to the generation of ROS is not clear. In quiescent cardiomyocytes, one study reports that the oxygen demands average 0.268 µl of O₂/min per 10⁵ myocytes (13), or 0.536 µl/min for the 2 x 10⁵ myocytes/well used in the present protocol. Total oxygen utilization is calculated to be 2.68 µl/well of 2 x 10⁵ cells over each 5-min postconditioning cycle. The oxygen available in each 5-ml well at 37°C with an average PO₂ of 40 mmHg during postconditioning is calculated to be 6.2 µl of O₂/5-ml well. Therefore, the oxygen available exceeds the ambient demands of the cardiomyocytes assuming that there is no replenishment from the incubator's hypoxic environment. Hence it is not likely that postconditioning limits the oxygen available for ROS generation in the present experimental conditions, although this hypothesis must be tested in appropriately designed experiments. In addition, there are no data as yet on whether postconditioning attenuates activity of the various oxidant-producing enzymes in cardiomyocytes. As an alternative mechanism, postconditioning may have preserved the intracellular antioxidant reserve that is sometimes depleted by in vivo ischemia-reperfusion (25) but less so in vitro (7). However, the endogenous antioxidant status (glutathione peroxidase, catalase) was not determined in the present study.

The present study shows that postconditioning attenuates ROS generated by cardiomyocytes independent of other sources of ROS such as endothelial cells and neutrophils. However, this study does not determine whether postconditioning reduces ROS by modulating the release of triggers such as endogenous adenosine (28), which has been reported to attenuate ROS generation (44) and salvage myocytes in hypoxic and ischemic conditions. Other factors such as nitric oxide may be involved in postconditioning (51), although it is not yet clear whether nitric oxide acts as a proximal trigger or instead acts distal to ATP-sensitive K⁺ channels. Postconditioning may also trigger survival-signaling pathways or inhibit proinjury pathways mediated by ERK1/2 (51), PKC, or phosphatidylinositol 3-kinase-Akt (46a, 50) that may be involved in protection during reperfusion.

The duration of postconditioning cycles (i.e., 5 min each) differed from that used in in vivo studies of coronary occlusion-reperfusion (29, 50, 51, 53). Indeed, the duration of each postconditioning cycle and the number of cycles applied have differed even between species in vivo (29, 52). The 5-min duration of postconditioning cycles used in the present in vitro study was determined after pilot studies were performed that used a range of cycle durations. One hypothesis for the differences in effective cycle duration involves the species differences in the metabolic rate of the myocardium, which is lower for cardiomyocytes compared with in vivo small- and large-animal working hearts. This difference in metabolic rate may be pertinent if postconditioning triggers or delays the release of endogenous autacoids (28) and may be important in determining the severity of the oxygen supply/demand mismatch that governs ROS generation during ischemia-reperfusion vs. hypoxia-reoxygenation. For example, hearts with a slow heart rate and low oxygen demand during ischemia (i.e., canine and rabbit) are postconditioned with longer period cycles (30 s) of reperfusion and ischemia compared with hearts with a faster heart rate (10-s period). A recent report supports this notion by showing that reperfusion-induced injury can be attenuated when a low metabolic rate organ such as liver is postconditioned by cycles of reperfusion-ischemia that last 2, 3, 5, and 7 min (46). The role of cycle duration in effectiveness of cardioprotection still requires further clarification.

In conclusion, the present study demonstrates for the first time that postconditioning reduces cell death in isolated neonatal rat cardiomyocytes, which is in agreement with the observed cardioprotection of in vivo models (29, 52). The reduction of the intracellular Ca²⁺ overload coincident with an attenuation of ROS generation and ROS-mediated lipid peroxidation suggests that there is a component of protection by postconditioning that is independent of inflammatory and neutrophil-endothelial cell interactions. However, these data do not rule out an active role played by neutrophils and their products in the in vivo setting. Additional studies are required to clarify the inhibitory mechanisms operative in attenuating noninflammatory and inflammatory cell-mediated processes. The data from the present study provide evidence to further support previous findings (29, 52) that the ischemic reperfused myocardium can be protected from reperfusion injury by intermittent reperfusion and ischemia at the time when blood supply is restored. Postconditioning is therefore yet another example of endogenous cardioprotection marshaled by the heart.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institutes of Health Grants HL-64886 (to Z-Q Zhao) and HL-69487 (to J. Vinten-Johansen) as well as funds from the Carlyle Fraser Heart Center of Emory University School of Medicine.

	ACKNOWLEDGMENTS

 
The authors are grateful for the technical contributions of Sara Katzmark and Susan Schmarkey to this study and for the assistance of Laurie Berley in preparing the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Z.-Q. Zhao, Cardiothoracic Research Laboratory, Carlyle Fraser Heart Center/Crawford Long Hospital, Emory Univ. School of Medicine, 550 Peachtree St. NE, Atlanta, GA 30308-2225 (E-mail: zzhao{at}emory.edu)

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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Akao M, O'Rourke B, Teshima Y, Seharaseyon J, and Marban E. Mechanistically distinct steps in the mitochondrial death pathway triggered by oxidative stress in cardiac myocytes. Circ Res 92: 186–194, 2003.[Abstract/Free Full Text]
2. Ambrosio G and Tritto I. Reperfusion injury: experimental evidence and clinical implications. Am Heart J 138: S69–S75, 1999.[CrossRef][Web of Science][Medline]
3. Bagchi D, Wetscher GJ, Bagchi M, Hinder PR, Perdikis G, Stohs SJ, Hinder RA, and Das DK. Interrelationship between cellular calcium homeostasis and free radical generation in myocardial reperfusion injury. Chem Biol Interact 104: 65–85, 1997.[CrossRef][Web of Science][Medline]
4. Bayfield MS, Lindner JR, Kaul S, Ismail S, Sheil ML, Goodman NC, Zacour R, and Spotnitz WD. Deoxygenated blood minimizes adherence of sonicated albumin microbubbles during cardioplegic arrest and after blood reperfusion: experimental and clinical observations with myocardial contrast echocardiography. J Thorac Cardiovasc Surg 113: 1100–1108, 1997.[Abstract/Free Full Text]
5. Berrizbeitia LD, McGrath LB, and Klabunde RE. Oxygen modulation of superoxide radical injury in the Krebs-perfused, isolated rabbit heart. J Invest Surg 15: 251–257, 2002.[CrossRef][Web of Science][Medline]
6. Braunwald E and Kloner RA. Myocardial reperfusion: a double-edged sword? J Clin Invest 76: 1713–1719, 1985.[Web of Science][Medline]
7. Coudray C, Boucher F, Pucheu S, de Leiris J, and Favier A. Relationship between severity of ischemia and oxidant scavenger enzyme activities in the isolated rat heart. Int J Biochem Cell Biol 27: 61–69, 1995.[CrossRef][Web of Science][Medline]
8. Crompton M and Andreeva L. On the involvement of a mitochondrial pore in reperfusion injury. Basic Res Cardiol 88: 513–523, 1993.[CrossRef][Web of Science][Medline]
9. Duilio C, Ambrosio G, Kuppusamy P, DiPaula A, Becker LC, and Zweier JL. Neutrophils are primary source of O₂ radicals during reperfusion after prolonged myocardial ischemia_. Am J Physiol Heart Circ Physiol 280: H2649–H2657, 2001.[Abstract/Free Full Text]
10. Duranteau J, Chandel NS, Kulisz A, Shao Z, and Schumacker PT. Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes. J Biol Chem 273: 11619–11624, 1998.[Abstract/Free Full Text]
11. Ermak G and Davies KJA. Calcium and oxidative stress: from cell signaling to cell death. Mol Immunol 38: 713–721, 2001.
12. Gen W, Tani M, Takeshita J, Ebihara Y, and Tamaki K. Mechanisms of Ca²⁺ overload induced by extracellular H₂₀₂ quiescent isolated rat cardiomyocytes. Basic Res Cardiol 96: 623–629, 2001.[CrossRef][Web of Science][Medline]
13. Gong GX, Weiss HR, Tse J, and Scholz PM. Exogenous nitric oxide reduces oxygen consumption of isolated ventricular myocytes less than other forms of guanylate cyclase stimulation. Eur J Pharmacol 344: 299–305, 1998.[CrossRef][Web of Science][Medline]
14. Grill HP, Zweier JL, Kuppusamy P, Weisfeldt ML, and Flaherty JT. Direct measurement of myocardial free radical generation in an in vivo model: effects of postischemic reperfusion and treatment with human recombinant superoxide dismutase. J Am Coll Cardiol 20: 1604–1611, 1992.[Abstract]
15. Guidarelli A, Brambilla L, Clementi E, Sciorati C, and Cantoni O. Stimulation of oxygen consumption promotes mitochondrial calcium accumulation, a process associated with, and causally linked to, enhanced formation of tert-butylhydroperoxide-induced DNA single-strand breaks. Exp Cell Res 237: 176–185, 1997.[CrossRef][Web of Science][Medline]
16. Halestrap AP, Clarke SJ, and Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion—a target for cardioprotection. Cardiovasc Res 61: 372–385, 2004.[Abstract/Free Full Text]
17. Halestrap AP, Kerr PM, Javadov S, and Woodfield KY. Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart. Biochim Biophys Acta 1366: 79–94, 1998.[Medline]
18. Halkos ME, Kerendi F, Corvera JS, Wang NP, Kin H, Payne CS, Sun HY, Guyton RA, Vinten-Johansen J, and Zhao ZQ. Myocardial protection with postconditioning is not enhanced by ischemic preconditioning. Ann Thorac Surg 78: 961–969, 2004.[Abstract/Free Full Text]
19. Hausenloy DJ, Duchen MR, and Yellon DM. Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion injury. Cardiovasc Res 60: 617–625, 2003.[Abstract/Free Full Text]
20. Hausenloy DJ, Maddock HL, Baxter GF, and Yellon DM. Inhibiting mitochondrial permeability transition pore opening: a new paradigm for myocardial preconditioning? Cardiovasc Res 55: 534–543, 2002.[Abstract/Free Full Text]
21. Hearse DJ. Reperfusion-induced injury: a possible role for oxidant stress and its manipulation. Cardiovasc Drugs Ther 5: 225–235, 1991.
22. Herzog WR, Vogel RA, Schlossberg ML, Edenbaum LR, Scott HJ, and Serebruany VL. Short-term low dose intracoronary diltiazem administered at the onset of reperfusion reduces myocardial infarct size. Int J Cardiol 59: 21–27, 1997.[CrossRef][Web of Science][Medline]
23. Igarashi-Saito K, Tsutsui H, Yamamoto S, Takahashi M, Kinugawa S, Tagawa H, Usui M, Yamamoto M, Egashira K, and Takeshita A. Role of SR Ca²⁺-ATPase in contractile dysfunction of myocytes in tachycardia-induced heart failure. Am J Physiol Heart Circ Physiol 275: H31–H40, 1998.[Abstract/Free Full Text]
24. Inoue T, Ku K, Kaneda T, Zang Z, Otaki M, and Oku H. Cardioprotective effects of lowering oxygen tension after aortic unclamping on cardiopulmonary bypass during coronary artery bypass grafting. Circ J 66: 718–722, 2002.[CrossRef][Web of Science][Medline]
25. Jones SP, Hoffmeyer MR, Sharp BR, Ho YS, and Lefer DJ. Role of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion. Am J Physiol Heart Circ Physiol 284: H277–H282, 2003.[Abstract/Free Full Text]
26. Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K, Antos CL, Olson EN, and Sollott SJ. Glycogen synthase kinase-3 mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 113: 1535–1549, 2004.[CrossRef][Web of Science][Medline]
27. Kevin LG, Camara AKS, Riess ML, Novalija E, and Stowe DF. Ischemic preconditioning alters real-time measure of O₂ radicals in intact hearts with ischemia and reperfusion. Am J Physiol Heart Circ Physiol 284: H566–H574, 2003.[Abstract/Free Full Text]
28. Kin H, Lofye MT, Amerson BS, Zatta AJ, Kerendi F, Halkos ME, Zhao ZQ, Headrick J, Guyton RA, and Vinten-Johansen J. Cardioprotection by "postconditioning" is mediated by increased retention of endogenous intravascular adenosine and activation of A_2a receptors during reperfusion (Abstract). Circulation 110: III-168, 2004.
29. Kin H, Zhao ZQ, Sun HY, Wang NP, Corvera JS, Halkos ME, Kerendi F, Guyton RA, and Vinten-Johansen J. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res 62: 74–85, 2004.[Abstract/Free Full Text]
30. Lefer DJ and Granger DN. Oxidative stress and cardiac disease. Am J Med 109: 315–323, 2000.[CrossRef][Web of Science][Medline]
31. Li C and Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 282: C227–C241, 2002.[Abstract/Free Full Text]
32. Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, Melendez JA, and Robinson JP. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem 278: 8516–8525, 2003.[Abstract/Free Full Text]
33. Lounsbury KM, Hu Q, and Ziegelstein RC. Calcium signaling and oxidant stress in the vasculature. Free Radic Biol Med 28: 1362–1369, 2000.[CrossRef][Web of Science][Medline]
34. Massoudy P, Mempel T, Raschke P, and Becker BF. Reduction of oxygen delivery during post-ischemic reperfusion protects the isolated guinea pig heart. Basic Res Cardiol 94: 231–237, 1999.[CrossRef][Web of Science][Medline]
35. Mukherjee SB, Das M, Sudhandiran G, and Shaha C. Increase in cytosolic Ca²⁺ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes. J Biol Chem 277: 24717–24727, 2002.[Abstract/Free Full Text]
36. Narayan P, Mentzer RM Jr, and Lasley RD. Annexin V staining during reperfusion detects cardiomyocytes with unique properties. Am J Physiol Heart Circ Physiol 281: H1931–H1937, 2001.[Abstract/Free Full Text]
37. Ng CKD, Deshpande SS, Irani K, and Alevriadou BR. Adhesion of flowing monocytes to hypoxia-reoxygenation-exposed endothelial cells: role of Rac1, ROS, and VCAM-1. Am J Physiol Cell Physiol 283: C93–C102, 2002.[Abstract/Free Full Text]
38. Opie LH. Role of calcium and other ions in reperfusion injury. Cardiovasc Drugs Ther 5: 237–247, 1991.
39. Piper HM, Garcia-Dorado D, and Ovize M. A fresh look at reperfusion injury. Cardiovasc Res 38: 291–300, 1998.[Free Full Text]
40. Przyklenk K, Darling CE, Dickson EW, and Wittaker P. Cardioprotection outside the box: the evolving paradigm of remote preconditioning. Basic Res Cardiol 98: 149–157, 2003.[Web of Science][Medline]
41. Rakhit RD, Mojet MH, Marber MS, and Duchen MR. Mitochondria as targets for nitric oxide-induced protection during simulated ischemia and reoxygenation in isolated neonatal cardiomyocytes. Circulation 103: 2617–2623, 2001.[Abstract/Free Full Text]
42. Rothstein EC, Byron KL, Reed RE, Fliegel L, and Lucchesi PA. H₂O₂-induced Ca²⁺ overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway. Am J Physiol Heart Circ Physiol 283: H598–H605, 2002.[Abstract/Free Full Text]
43. Sharikabad MN, Ostbye KM, and Brors O. Increased [Mg²⁺]_o reduces Ca²⁺ influx and disruption of mitochondrial membrane potential during reoxygenation. Am J Physiol Heart Circ Physiol 281: H2113–H2123, 2001.[Abstract/Free Full Text]
44. Sohn HY, Krotz F, Gloe T, Keller M, Theisen K, Klauss V, and Pohl U. Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine. Cardiovasc Res 58: 638–646, 2003.[Abstract/Free Full Text]
45. Sun HY, Wang NP, Halkos ME, Kerendi F, Kin H, Wang RX, Guyton RA, and Zhao ZQ. Involvement of Na⁺/H⁺ exchanger in hypoxia/re-oxygenation-induced neonatal rat cardiomyocyte apoptosis. Eur J Pharmacol 486: 121–131, 2004.[CrossRef][Web of Science][Medline]
46. Sun K, Liu ZS, and Sun Q. Role of mitochondria in cell apoptosis during hepatic ischemia-reperfusion injury and protective effect of ischemic postconditioning. World J Gastroenterol 10: 1934–1938, 2004.[Medline]
46. Tsang A, Hausenloy DJ, Mocanu MM, and Yellon DM. Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ Res 95: 230–232, 2004.[Abstract/Free Full Text]
47. Vinten-Johansen J. Reperfusion injury: idle curiosity or therapeutic vector? J Thromb Thrombolysis 4: 59–61, 1997.[CrossRef][Medline]
48. Vinten-Johansen J. Involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury. Cardiovasc Res 61: 481–497, 2004.[Abstract/Free Full Text]
49. Waypa GB, Marks JD, Mack MM, Boriboun C, Mungai PT, and Schumacker PT. Mitochondrial reactive oxygen species trigger calcium increases during hypoxia in pulmonary arterial myocytes. Circ Res 91: 719–726, 2002.[Abstract/Free Full Text]
50. Yang XM, Philipp S, Downey JM, and Cohen MV. Postconditioning's protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation. Basic Res Cardiol 100: 57–63, 2005.[CrossRef][Web of Science][Medline]
51. Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, and Cohen MV. Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardiol 44: 1103–1110, 2004.[Abstract/Free Full Text]
52. Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, and Vinten-Johansen J. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 285: H579–H588, 2003.[Abstract/Free Full Text]
53. Zhao ZQ, Corvera JS, Wang NP, Guyton RA, and Vinten-Johansen J. Reduction in infarct size and preservation of endothelial function by ischemic postconditioning: comparison with ischemic preconditioning (Abstract). Circulation 106, Suppl: II-314, 2002.
54. Zhou X, Zhao X, and Ashraf M. Direct evidence that initial oxidative stress triggered by preconditioning contributes to second window of protection by endogenous antioxidant enzyme in myocytes. Circulation 93: 1177–1184, 1996.[Abstract/Free Full Text]
55. Zweier JL, Wang P, and Kuppusamy P. Direct measurement of nitric oxide generation in the ischemic heart using electron paramagnetic resonance spectroscopy. J Biol Chem 270: 304–307, 1995.[Abstract/Free Full Text]

This article has been cited by other articles:

				V. Pialoux, P. J. Hanly, G. E. Foster, J. V. Brugniaux, A. E. Beaudin, S. E. Hartmann, M. Pun, C. T. Duggan, and M. J. Poulin Effects of Exposure to Intermittent Hypoxia on Oxidative Stress and Acute Hypoxic Ventilatory Response in Humans Am. J. Respir. Crit. Care Med., November 15, 2009; 180(10): 1002 - 1009. [Abstract] [Full Text] [PDF]

				C. Leconte, E. Tixier, T. Freret, J. Toutain, R. Saulnier, M. Boulouard, S. Roussel, P. Schumann-Bard, and M. Bernaudin Delayed Hypoxic Postconditioning Protects Against Cerebral Ischemia in the Mouse Stroke, October 1, 2009; 40(10): 3349 - 3355. [Abstract] [Full Text] [PDF]

				A. Granfeldt, D. J. Lefer, and J. Vinten-Johansen Protective ischaemia in patients: preconditioning and postconditioning Cardiovasc Res, July 15, 2009; 83(2): 234 - 246. [Abstract] [Full Text] [PDF]

				I. Szwarc, G. Mourad, and A. Argiles Post-conditioning to reduce renal ischaemia/reperfusion injury Nephrol. Dial. Transplant., July 1, 2009; 24(7): 2288 - 2289. [Full Text] [PDF]

				U. Abdel-Rahman, P. Risteski, K. Tizi, S. Kerscher, S. Bejati, K. Zwicker, M. Scholz, U. Brandt, and A. Moritz Hypoxic reoxygenation during initial reperfusion attenuates cardiac dysfunction and limits ischemia-reperfusion injury after cardioplegic arrest in a porcine model J. Thorac. Cardiovasc. Surg., April 1, 2009; 137(4): 978 - 982. [Abstract] [Full Text] [PDF]

				Q. C. Yong, S. W. Lee, C. S. Foo, K. L. Neo, X. Chen, and J.-S. Bian Endogenous hydrogen sulphide mediates the cardioprotection induced by ischemic postconditioning Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1330 - H1340. [Abstract] [Full Text] [PDF]

				S. E. McAllister, H. Ashrafpour, N. Cahoon, N. Huang, M. A. Moses, P. C. Neligan, C. R. Forrest, J. E. Lipa, and C. Y. Pang Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R681 - R689. [Abstract] [Full Text] [PDF]

				J.-y. Wang, J. Shen, Q. Gao, Z.-g. Ye, S.-y. Yang, H.-w. Liang, I. C. Bruce, B.-y. Luo, and Q. Xia Ischemic Postconditioning Protects Against Global Cerebral Ischemia/Reperfusion-Induced Injury in Rats Stroke, March 1, 2008; 39(3): 983 - 990. [Abstract] [Full Text] [PDF]

				L. Argaud, O. Gateau-Roesch, L. Augeul, E. Couture-Lepetit, J. Loufouat, L. Gomez, D. Robert, and M. Ovize Increased mitochondrial calcium coexists with decreased reperfusion injury in postconditioned (but not preconditioned) hearts Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H386 - H391. [Abstract] [Full Text] [PDF]

				R. Jiang, A. Zatta, H. Kin, N. Wang, J. G. Reeves, J. Mykytenko, J. Deneve, Z.-Q. Zhao, R. A. Guyton, and J. Vinten-Johansen PAR-2 activation at the time of reperfusion salvages myocardium via an ERK1/2 pathway in in vivo rat hearts Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2845 - H2852. [Abstract] [Full Text] [PDF]

				M. Fujita, H. Asanuma, A. Hirata, M. Wakeno, H. Takahama, H. Sasaki, J. Kim, S. Takashima, O. Tsukamoto, T. Minamino, et al. Prolonged transient acidosis during early reperfusion contributes to the cardioprotective effects of postconditioning Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H2004 - H2008. [Abstract] [Full Text] [PDF]

				Q. Chen, A. K. S. Camara, D. F. Stowe, C. L. Hoppel, and E. J. Lesnefsky Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion Am J Physiol Cell Physiol, January 1, 2007; 292(1): C137 - C147. [Abstract] [Full Text] [PDF]

				N. Couvreur, L. Lucats, R. Tissier, A. Bize, A. Berdeaux, and B. Ghaleh Differential effects of postconditioning on myocardial stunning and infarction: a study in conscious dogs and anesthetized rabbits Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1345 - H1350. [Abstract] [Full Text] [PDF]

				M. Schmelter, B. Ateghang, S. Helmig, M. Wartenberg, and H. Sauer Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation FASEB J, June 1, 2006; 20(8): 1182 - 1184. [Abstract] [Full Text] [PDF]

				O. Gateau-Roesch, L. Argaud, and M. Ovize Mitochondrial permeability transition pore and postconditioning Cardiovasc Res, May 1, 2006; 70(2): 264 - 273. [Abstract] [Full Text] [PDF]

				A. J. Zatta, H. Kin, G. Lee, N. Wang, R. Jiang, R. Lust, J. G. Reeves, J. Mykytenko, R. A. Guyton, Z.-Q. Zhao, et al. Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling Cardiovasc Res, May 1, 2006; 70(2): 315 - 324. [Abstract] [Full Text] [PDF]

				L. M. Schwartz and C. J. Lagranha Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1011 - H1018. [Abstract] [Full Text] [PDF]

				S. P. Loukogeorgakis, A. T. Panagiotidou, D. M. Yellon, J. E. Deanfield, and R. J. MacAllister Postconditioning Protects Against Endothelial Ischemia-Reperfusion Injury in the Human Forearm Circulation, February 21, 2006; 113(7): 1015 - 1019. [Abstract] [Full Text] [PDF]

				F. Dong, X. Zhang, and J. Ren Leptin Regulates Cardiomyocyte Contractile Function Through Endothelin-1 Receptor-NADPH Oxidase Pathway Hypertension, February 1, 2006; 47(2): 222 - 229. [Abstract] [Full Text] [PDF]

				C. E. Darling, R. Jiang, M. Maynard, P. Whittaker, J. Vinten-Johansen, and K. Przyklenk Postconditioning via stuttering reperfusion limits myocardial infarct size in rabbit hearts: role of ERK1/2 Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1618 - H1626. [Abstract] [Full Text] [PDF]

				A. Tsang, D. J. Hausenloy, and D. M. Yellon Myocardial postconditioning: reperfusion injury revisited Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H2 - H7. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 288/4/H1900    most recent 01244.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (47) Citing Articles via Google Scholar Google Scholar Articles by Sun, H.-Y. Articles by Zhao, Z.-Q. Search for Related Content PubMed PubMed Citation Articles by Sun, H.-Y. Articles by Zhao, Z.-Q.