Cardiac preconditioning, including that induced by halogenated anesthetics, is an innate protective mechanism against ischemia-reperfusion injury. The adenosine triphosphate-sensitive potassium (K(ATP)) channels are considered essential in preconditioning mechanism. However, it is unclear whether K(ATP) channels are triggers initiating the preconditioning signaling, and/or effectors responsible for the cardioprotective memory and activated during ischemia-reperfusion.
Adult rat cardiomyocytes were exposed to oxidative stress with 200 microM H(2)O(2) and 100 microM FeSO4. Myocyte survival was determined based on morphologic characteristics and trypan blue exclusion. To induce preconditioning, the myocytes were pretreated with isoflurane. The involvement of sarcolemmal and mitochondrial K(ATP) channels was investigated using specific inhibitors HMR-1098 and 5-hydroxydecanoic acid. Data are expressed as mean +/- SD.
Oxidative stress induced cell death in 47 +/- 14% of myocytes. Pretreatment with isoflurane attenuated this effect to 26 +/- 8%. Blockade of the sarcolemmal K(ATP) channels abolished the protection by isoflurane pretreatment when HMR-1098 was applied throughout the experiment (50 +/- 21%) or only during oxidative stress (50 +/- 12%), but not when applied during isoflurane pretreatment (29 +/- 13%). Inhibition of the mitochondrial K(ATP) channels abolished cardioprotection irrespective of the timing of 5-hydroxydecanoic acid application. Cell death was 42 +/- 23, 45 +/- 23, and 46 +/- 22% when 5-hydroxydecanoic acid was applied throughout the experiment, only during isoflurane pretreatment, or only during oxidative stress, respectively.
The authors conclude that both sarcolemmal and mitochondrial K(ATP) channels play essential and distinct roles in protection afforded by isoflurane. Sarcolemmal K(ATP) channel seems to act as an effector of preconditioning, whereas mitochondrial K(ATP) channel plays a dual role as a trigger and an effector.
CARDIAC preconditioning is an innate protective mechanism against injury by ischemia and reperfusion.1In addition to the extent of cardioprotection, one of the most remarkable characteristics of preconditioning is the memory phase, when the cardioprotective effects persist despite removal of the preconditioning stimulus. To date, numerous studies have investigated the mechanism of preconditioning, and many crucial components underlying cardioprotection have been identified. Adenosine triphosphate-sensitive potassium (KATP) channels have long been considered essential components of cardioprotection by ischemic and pharmacologic preconditioning. Acting as metabolic sensors, regulated by intracellular metabolic factors such as adenosine triphosphate, adenosine diphosphate, and cytosolic pH, they are attractive candidates as the major contributors to the mechanism of cardioprotection. Their critical role for cardiac preconditioning, including that induced by halogenated anesthetics, has been demonstrated in a number of studies.2–5
There are two populations of KATPchannels in cardiac myocytes: the mitochondrial (mitoKATP) channel located in the inner mitochondrial membrane and the sarcolemmal (sarcKATP) channel located in the plasma membrane. Initially, before discovery of mitoKATPchannels, the cardioprotective effects of preconditioning were attributed to the sarcKATPchannels.2The protective effects of KATPchannel opening were ascribed to action potential shortening and the resulting decrease in Ca2+overload during ischemia and reperfusion.6However, later studies demonstrated that cardioprotective actions of KATPchannel openers are independent of the action potential shortening.7,8After a more recent discovery of the mitoKATPchannels in the inner mitochondrial membrane and development of selective mito and sarcKATPchannel inhibitors, evidence suggested that mitoKATPchannels rather than the sarcKATPchannels play a more important role in cardioprotection.9,10HMR-1098, a specific inhibitor of sarcKATPchannel, has often failed to abolish protection by ischemic and pharmacologic preconditioning, whereas cardioprotection was elicited by the mitoKATPchannel activator diazoxide.4,10This led to a widespread opinion that cardioprotective effects of preconditioning depend mostly on the mitoKATPchannel. However, some of the recent studies that involved the use of sarcKATP-specific inhibitors and genetic models of disrupted or knocked-out sarcKATPchannel subunits indicated that the role of sarcKATPchannel in cardioprotection should not be ignored.11–13
The involvement of sarcKATPchannels in cardioprotection by anesthetic-induced preconditioning (APC) was demonstrated in a limited number of studies.14,15More studies demonstrated a predominant role of the mitoKATPchannels in APC, while finding no apparent involvement of the sarcKATPchannel.16–19In addition to the conflicting results regarding the relative importance of sarcKATPand mitoKATPchannels, their specific roles remain unclear. It is uncertain whether they are triggers, important for initiating the preconditioning signaling cascade, or effectors, the endpoints of the preconditioning cascade, responsible for the cardioprotection memory and activated during ischemia-reperfusion.4,5,20
Therefore, the goal of the current study was to test whether sarcKATPand mitoKATPchannels contribute to isoflurane preconditioning and to investigate their exact role in protection afforded by isoflurane. We found that both sarcKATPand mitoKATPchannels are essential components of APC and that each channel plays a distinct role: The sarcKATPchannel acts as an effector, whereas the mitoKATPchannel acts both as a trigger and as an effector of preconditioning.
Materials and Methods
The animal use and experimental protocols of this study were approved by the Animal Use and Care Committee of the Medical College of Wisconsin, Milwaukee, Wisconsin.
Ventricular myocytes were isolated from hearts of adult male Wistar rats (150–250 g) by enzymatic dissociation. The rats were injected with heparin (1,000 U intraperitoneally) and anesthetized with thiobutabarbital (Inactin, 100 mg/kg intraperitoneally; Sigma-Aldrich, St. Louis, MO). After thoracotomy, the hearts were excised, mounted on a Langendorff apparatus, and retrogradely perfused with heparinized Joklik medium (Gibco BRI; Invitrogen, Grand Island, NY) at the flow rate of 7 ml/min. After the blood had been washed out, the perfusate was replaced with an enzyme solution containing Joklik medium supplemented with 0.5 mg/ml collagenase type II (Invitrogen, Carlsberg, CA), 0.25 mg/ml protease XIV (Sigma-Aldrich), and 1 mg/ml bovine serum albumin (Serologicals, Kankakee, IL) at pH 7.23. All solutions were continuously gassed with a mixture of 95% O2–5% CO2and were kept at 37°C. After 25 min of enzyme digestion, the ventricles were excised, minced, and incubated in the enzyme solution for additional 8–10 min in a shaker bath at 37°C. The cell suspension was filtered through 200 μm mesh and centrifuged. The cell pellet was then washed twice in modified Tyrode solution (132 mm NaCl, 10 mm HEPES, 5 mm glucose, 5 mm KCl, 1 mm CaCl2, and 1.2 mm MgCl2, adjusted to pH 7.4). After isolation, the myocytes were stored in Tyrode solution at room temperature and allowed to recover for 1 h before the cell survival experiments. All experiments were performed within 5 h after the cell isolation procedure.
The suspension of isolated cardiomyocytes (1 ml) was placed in a chamber on the stage of inverted microscope (Diaphot 300; Nikon, Tokyo, Japan), and cells were allowed to settle for 10 min. The myocytes were then stained with 1 ml trypan blue solution, 0.4% (Sigma-Aldrich), for 2 min followed by a dye washout with glucose-free Tyrode solution (132 mm NaCl, 10 mm HEPES, 5 mm KCl, 1 mm CaCl2, and 1.2 mm MgCl2, adjusted to pH 7.4). Cells that were rod-shaped and excluded trypan blue were considered living21and were counted. The counting time was monitored and was kept uniform in all experiments (10 min). In each experiment, approximately 250 myocytes were counted. After cell counting, perfusion with glucose-free Tyrode was started. Thirty minutes into the experiment, the myocytes were exposed to oxidative stress by perfusion with 200 μm H2O2and 100 μm FeSO4·7H2O (both from Sigma-Aldrich) for 17 min. The mixture of H2O2and Fe2+yields a highly reactive hydroxyl radical (OH·) via the Fenton reaction.22The 17-min duration was chosen because it was optimal for damaging approximately 50% of myocytes. After oxidative stress, the myocytes were reperfused with glucose-free Tyrode solution for another 20 min and stained with trypan blue, after which the remaining living cells were counted. The percentage of cell death was calculated from the cell count before and after oxidative stress. The location of the myocytes was monitored using a chamber bottom with a labeled grid. This enabled counting of the same myocytes before and after oxidative stress.
The time between cell counts before and after stress was exactly 67 min in all experiments. In the experimental groups that underwent APC, the myocytes were exposed to isoflurane for 20 min and 5 min anesthetic washout before oxidative stress. To investigate the effects of the sarcKATPand mitoKATPchannels on cell survival, the specific inhibitor HMR-1098 (50 μm; a gift from Aventis Pharma, Frankfurt am Main, Germany) or 5-hydroxydecanoic acid (5-HD, 200 μm; Sigma-Aldrich, St. Louis, MO) was added to the superfusing solution at different time points. In all experimental groups, n indicates the number of animals. The protocols for all experimental groups are illustrated in figure 1.
Isoflurane (Abbott Laboratories, North Chicago, IL) was dispersed in glucose-free Tyrode solution by sonication and delivered to cardiomyocytes from the airtight glass syringes. At the end of each experiment, samples of isoflurane-containing solution were collected from the chamber, and the concentrations of isoflurane were analyzed by gas chromatography (Shimadzu, Kyoto, Japan). The average concentration of isoflurane used in this study was 0.51 ± 0.09 mm, equivalent to 1.2 vol% at 22°C. The 5-min washout period was sufficient to remove all isoflurane from the chamber as confirmed by isoflurane measurements after the washout. HMR-1098 and 5-HD were kept as stock solution in double-distilled water. All stock solutions were diluted to required concentration in the superfusing buffer immediately before application.
Results are expressed as mean ± SD. Data were analyzed using Origin 7 software (OriginLab, Northampton, MA). Statistical analysis was performed using one-way analysis of variance with Scheffépost hoc test. Differences were considered significant when the two-tailed P value was less than 0.05.
Isoflurane Protects Isolated Cardiomyocytes from Oxidative Stress
Oxidative stress was used to mimic the reperfusion injury and investigate isoflurane-induced myocyte protection. In the time control group, 67 min of perfusion with glucose-free Tyrode solution had no significant effect on cell death (7 ± 5%, n = 5; fig. 2, TC group). During exposure to 200 μm H2O2and 100 μm FeSO4, some of the otherwise nonbeating cardiomyocytes started contracting, which resulted in cell hypercontracture and death in 47 ± 14% of cardiomyocytes (n = 11, stress group). When cardiomyocytes were pretreated with isoflurane before oxidative stress (APC group), cell death was markedly attenuated to 26 ± 8% (P < 0.05, n = 10). These results demonstrated that in vitro pretreatment with isoflurane protects the cardiomyocytes from damage by oxidative stress.
Sarcolemmal KATPChannel Blockade during Oxidative Stress, but Not during Isoflurane Pretreatment, Abolishes Protection by Isoflurane
To investigate the role of sarcKATPchannels in protection by isoflurane, the selective sarcKATPchannel inhibitor HMR-1098 was used. HMR-1098 (50 μm) applied throughout the experiment had no effect on myocyte death in the time control group (5 ± 3%, n = 6; fig. 3A, TC + HMR group). Application of HMR-1098 did not potentiate the deleterious effects of oxidative stress, and cell damage in this group was 48 ± 13% (n = 9; fig. 3A, stress + HMR group). However, inhibition of the sarcKATPchannel completely abolished the protective effects of isoflurane, and cell death after isoflurane pretreatment in the presence of HMR-1098 was 50 ± 21% (n = 9; APC + HMR group).
To determine the exact time period in which the sarcKATPchannel activation is protective, HMR-1098 was applied only during isoflurane pretreatment or only during stress. When sarcKATPchannel inhibitor was applied during isoflurane exposure, the cardioprotective effects of isoflurane were still present and cell damage by oxidative stress was lower than without isoflurane pretreatment (29 ± 13%, n = 7; fig. 3B, APC + HMRISOgroup). However, when HMR-1098 was applied only during oxidative stress, the protective effects of isoflurane were completely abolished and cell death was 50 ± 12% (n = 7; APC + HMRstressgroup). These results show that activation of the sarcKATPchannel during the stress period is necessary for isoflurane-induced myocyte protection.
Mitochondrial KATPChannel Inhibition during Both Isoflurane Pretreatment and Oxidative Stress Abolishes Isoflurane-induced Protection
To assess contribution of the mitoKATPchannel to isoflurane-induced protection against oxidative stress, 5-HD (200 μm), a selective inhibitor of the mitoKATPchannels, was used. 5-HD was applied either throughout the experiment or during specific parts of the experiment as described previously. 5-HD did not affect cell damage in the time control group (7 ± 4%, n = 6; fig. 4A, TC + 5-HD group). In addition, inhibition of mitoKATPchannel had no effect on myocyte damage by oxidative stress, and cell death in the stress + 5-HD group was 44 ± 17% (n = 9; fig. 4A). However, when applied throughout the experiment, 5-HD completely abolished cellular protection by isoflurane, and cell death in APC + 5-HD group was 42 ± 23% (n = 8). When 5-HD was included during only the isoflurane exposure or only the stress period, cell death was 45 ± 23% and 46 ± 22% in the APC + 5-HDAPCand APC + HDstressgroups, respectively (both n = 6; fig. 4B). These results indicate that activation of the mitoKATPchannel during both isoflurane pretreatment and oxidative stress plays a role in protection by isoflurane.
In this study, freshly isolated adult cardiomyocytes were used to investigate specific roles of sarcKATPand mitoKATPchannels in cardioprotective effects of APC. It was found that both sarcKATPand mitoKATPchannels are essential for the protection of myocytes from damage by oxidative stress because their inhibition completely abolished the protective effects of isoflurane preconditioning. Specifically, activation of the sarcKATPchannel was required for the cardioprotective effects of APC during the stress period but not during the preconditioning period. In contrast, activation of mitoKATPchannel was found to be necessary during both isoflurane preconditioning and during exposure to oxidative stress. From these results, it seems that although activation of both channels is important for the cardioprotective effects of preconditioning, each channel plays a distinct role: The sarcKATPchannel acts as an effector, whereas the mitoKATPchannel plays a dual role as a trigger and an effector.
In our study, we found that isoflurane protects isolated cardiomyocytes from oxidative stress via both sarcKATPand mitoKATPchannel activation, because channel inhibition by HMR-1098 and 5-HD, respectively, abolished cardioprotective effects of APC. Similar results were reported by Toller et al .,14who demonstrated in a dog model in vivo that infarct size reduction achieved by desflurane preconditioning is abolished in the presence of HMR-1098 and 5-HD. Further, using the same inhibitors, both channels were found to mediate ketamine-induced protection of the force of contraction of human right atrial trabeculae during hypoxia and reoxygenation.15In contrast to these studies that demonstrated an equally important role for both sarcKATPand mitoKATPchannels, there are studies that demonstrate a more significant role of the mitoKATPchannels. For example, Zaugg et al .16found that mitoKATPchannel inhibition by 5-HD, but not sarcKATPchannel inhibition by HMR-1098, completely abolished the cardioprotective effects of isoflurane and sevoflurane in isolated adult rat cardiomyocytes exposed to ischemia. Similarly, Uecker et al .17showed that 5-HD completely blocked the cardioprotection by ischemic or isoflurane-induced preconditioning in isolated perfused rat hearts, whereas HMR-1098 had no effect. MitoKATPactivation was found to be essential, whereas sarcKATPchannel was found to play no role or have only a partial role in desflurane- and sevoflurane-induced preconditioning of isolated human right atria, respectively.18,19
Pronounced differences in findings from these studies that investigated the role of the sarcKATPchannel in APC may result from differences in the experimental models used (isolated cardiomyocytes, isolated hearts, atrial trabeculae, in vivo preparation). There are also differences in the insults on the heart (ischemia and reperfusion in vivo , simulated ischemia without reperfusion, hypoxia, oxidative stress, metabolic inhibition). Moreover, the measured endpoints of cardiac injury also differ (cell death and infarcted area vs . functional parameters such as developed force of contraction). Finally, there are differences in the time and duration of application of pharmacologic inhibitors (application during preconditioning stimulus vs . stress period). In our preparation, the timing of application of HMR-1098, but not of 5-HD, was found to be crucial. If HMR-1098 was applied only during the preconditioning stimulus period (isoflurane exposure), cytoprotection was still present, but when HMR-1098 was applied during oxidative stress, cytoprotection was abolished. Therefore, in evaluating the studies that test the role of the sarcKATPchannel in preconditioning, it is important to consider the timing of application of the inhibitors. Further, the endpoint variable used to evaluate the myocardial injury is also an important factor. For example, the sarcKATPchannel was found not to have a role in the infarct size reduction by adenosine-enhanced ischemic preconditioning, but it was important for the improvement of functional recovery.23In the same study, the mitoKATPchannel had effect primarily on the infarct size without affecting the functional recovery. Another important condition is the type of insult. Both sarc and mitoKATPchannels were shown to have distinct roles in ischemia-reperfusion injury. Activation of the mitoKATPchannel mediates the phorbol 12-myristate 13-acetate-induced protection against cell death during chemically induced hypoxia, but not during reoxygenation.11In contrast, activation of the sarcKATPchannel was protective only during reoxygenation.11Therefore, it is possible that if only hypoxia is used as an insult,16involvement of the sarcKATPchannel in protection against ischemia-reperfusion injury can be overlooked. In addition, the effectiveness of the inhibitors can be altered during application of certain insults. For example, HMR-1098 was found to be ineffective in blocking the sarcKATPcurrents during metabolic inhibition by NaCN and iodoacetate.24This may also explain the often reported lack of HMR-1098 effects on the cardioprotection by ischemic and pharmacologic preconditioning.
One of the most remarkable characteristics of the preconditioning phenomenon is the memory phase, when cardioprotection persists despite withdrawal of the preconditioning stimulus (such as anesthetic in APC). In the complex mechanism of preconditioning, one key element of the preconditioning pathway is triggers, activated during preconditioning stimulus and responsible for initiating the downstream signaling cascade. Another key component is the effectors, which are at the end of the preconditioning signaling pathway and are directly responsible for cardioprotective effects of preconditioning during the memory phase. Some of the identified triggers are Gi-coupled receptors, such as adenosine receptors and a small burst of reactive oxygen species.10,25The downstream signaling cascade includes activation of intracellular kinases, with protein kinase C playing a central role. Kinase activation results in phosphorylation and priming of the effectors and may potentially result in establishing the cardioprotection memory.26Both sarcKATPand mitoKATPchannels have been implicated as triggers and effectors of preconditioning, but studies have yielded controversial results. In our study, application of HMR-1098 during the stress phase, but not during the preconditioning phase, completely abrogated cardioprotective effects of APC. This suggests that the sarcKATPchannel acts as an effector, but not as a trigger of APC, being activated and acting protectively only during the memory phase. These results support our previous studies, which demonstrated that opening of the sarcKATPchannel is potentiated during the memory phase of APC and that protein kinase C-δ mediates this effect.27In the current study, application of 5-HD during both the preconditioning phase and the stress phase reversed cardioprotective effects of APC to the same extent, indicating that mitoKATPchannel acts as both trigger and effector. The findings were similar to those of Fryer et al .,28who demonstrated that the mitoKATPchannel acts as both trigger and effector of preconditioning by ischemia, because 5-HD administration before or after the preconditioning phase completely abolished cardioprotection in the rat hearts. In contrast, Pain et al .29demonstrated that the mitoKATPchannel acts only as a trigger of ischemic preconditioning in isolated rabbit hearts. Similarly, the mitoKATPchannel opening was found to trigger isoflurane-induced preconditioning of rabbit hearts by generating reactive oxygen species.30Interestingly, the mitoKATPchannel was found to be an effector, but not a trigger, whereas the sarcKATPchannel was shown to act as a trigger of the delayed cardioprotection by opioid-induced preconditioning in rats.31Furthermore, the role of the sarcKATPchannel as a trigger was demonstrated in the delayed ischemic preconditioning of the rat heart.32However, these studies investigated the delayed phase of preconditioning 24 h after the preconditioning stimulus, whereas our study was focused on the early phase of preconditioning.
The role of the mitoKATPchannel as both a trigger and an effector and the role of the sarcKATPchannel as the effector of preconditioning could be explained by the following sequence of events. Exposure to isoflurane can directly activate mitoKATPchannels.33Opening of the mitoKATPchannel results in changes in the mitochondrial bioenergetics, which may result in a small burst of reactive oxygen species,29that can further affect mitochondrial bioenergetics and activate cytosolic mediators such as protein kinase C.34–37Activated protein kinase C translocates to the sarcolemma and can then phosphorylate the sarcKATPchannel and sensitize it to opening.27,38The primed sarcKATPchannel opens sooner and/or more during subsequent metabolic stress (ischemia-reperfusion, oxidative stress, metabolic inhibition, and others), resulting in greater K+efflux, a more rapid repolarization of the cell membrane, and action potential shortening with subsequent contractile failure, which may thereby decrease cytosolic Ca2+loading during ischemia-reperfusion.12,13,39A similar sequence of events occurs in quiescent cardiomyocytes, where sarcKATPchannel opening prevents diastolic depolarization of the cell membrane and decreases Ca2+loading.40This can reduce or prevent mitochondrial Ca2+overload, a major trigger of the cell death pathway.41The mitoKATPchannel can also open during ischemia-reperfusion, which may result in depolarization of the inner mitochondrial membrane and thus further decrease driving force for the mitochondrial Ca2+entry and loading during ischemia-reperfusion.42Evidence that pretreatment with the mitoKATPchannel opener diazoxide causes early activation of the sarcKATPchannel during metabolic inhibition and protects the cells from Ca2+loading via indirect mechanism supports in part such a sequence of events.39From ours as well as other studies, it seems that both the sarcKATPand mitoKATPchannel are crucial components of cardioprotection that can interact and potentiate each other's protective effects. For example, Tanno et al .43showed that HMR-1098 can partially block the protection of isolated rabbit hearts from ischemia-reperfusion injury induced by diazoxide. In the same study, 5-HD completely abolished the cardioprotective effects of pretreatment with a low-dose pinacidil (10 μm) that is considered to open primarily sarcKATPchannels but not mitoKATPchannels.
It is important to acknowledge some limitations of our study. This study relies on specificity of pharmacologic openers and blockers of the sarcKATPand mitoKATPchannels. For example, both 5-HD and diazoxide were shown to have the mitoKATPchannel-independent effects in mitochondria,44and we cannot exclude the possibility that abolishment of APC by 5-HD in our study could have been due to the nonspecific effects. In addition, certain limitations are inherent to all in vitro models, including the isolated myocyte model. Disaggregated myocytes are in an artificial environment that is different from that in the whole organ, and in vitro conditions cannot replicate all of the complexity of the in vivo setting. However, one advantage of our model, which contains only myocytes without the presence of other cell types, is that we are able to isolate the effects of drugs (isoflurane, the channel inhibitors) on the cardiomyocytes without vascular and neuronal influences. Also, although the use of oxidative stress does not strictly mimic the conditions during ischemia-reperfusion, both H2O2and OH· were shown to be a relevant component of ischemia-reperfusion injury in vivo . Therefore, keeping in mind the limitations of the isolated myocyte model, this model might still provide information relevant to the in vivo setting.
From our results, we conclude that both the sarcKATPand the mitoKATPchannel play essential and distinct roles in APC in rat heart. The sarcKATPchannel acts as an effector of anesthetic preconditioning, whereas the mitoKATPchannel plays a dual role as a trigger and an effector.
The authors give special thanks to Martin Bienengraeber, Ph.D. (Assistant Professor, Departments of Anesthesiology and Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin), for helpful discussions. The authors also thank Mary Ziebell (Research Technologist, Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin) for isoflurane measurements.