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This review focuses on the role of reactive oxygen species in the pathogenesis of cardiac adaptation to ischemia. Results from various laboratories including the authors’ confirm the assumption that reactive oxygen species can be an integral part of the induction of both early and delayed forms of cardioprotection. There is conclusive evidence that reactive oxygen species may lead to activation of protein kinase C, protein tyrosine kinase, ionic channel openings and activation of transcriptional factors, all of which may translate into cardioprotection. These findings unveil a contradictory yet fascinating concept that oxygen radicals, although well recognized for their toxicity, can, under special circumstances, beneficially alter cell function leading to increased cell tolerance and survival.
Minimizing myocardial damage due to severe ischemia is of major concern in the therapy of patients suffering from ischemic heart disease. As a result of prolonged coronary occlusion, as in acute infarction, or during cardiopulmonary bypass, such patients are at risk of sustaining irreversible myocardial damage leading to depressed cardiac function. Thus, establishing early reperfusion has become a prerequisite in curbing ischemic injury to the heart muscle. However, such a measure is not entirely without its drawbacks, which is why reperfusing ischemic myocardium has been referred to as a ‘double-edged sword’ (1). Namely concurrent with postischemic reperfusion, one is inevitably confronted with reperfusion injury.
One theory behind reperfusion injury defines a central role for reactive oxygen species (ROS) (2). ROS are highly unstable molecules capable of attacking cell membrane and subcellular structures, leading to metabolic and structural changes that can be detrimental to cell survival (3). They include superoxide anion, hydrogen peroxide and hydroxyl radical, as well as many other active metabolites. It has been shown that concurrent with early reperfusion there is a burst in production of ROS. It is proposed that the xanthine oxidase system, activated neutrophils, the mitochondrial electron transport chain and the arachidonic acid pathway are potential sources for ROS generation (3,4). Thus, abundant data exist to postulate a detrimental role played by ROS during postischemic reperfusion, yet to elaborate further is beyond the scope of this review. More recently, however, it was shown that at low concentrations ROS can modulate functions within the cell. One such phenomenon where ROS are argued to have a positive impact on myocardial performance is the induction of cardiac adaptation to ischemia, otherwise known as ‘ischemic preconditioning’.
Ischemic preconditioning, first described by Murry et al (5), is the phenomenon of increased adaptive myocardial tolerance to severe ischemic insult following a short burst of nonlethal ischemia-reperfusion cycles. Murry and his coworkers had for years been following the relation between ATP loss during ischemia and eventual cell necrosis. At first they showed that in canine myocardium subjected to repeated short bursts of ischemia, preservation of ATP concentration during subsequent occlusions exceeded the rate of depletion during the first occlusion (6). In an extension of these findings they subjected animals to four cycles of 5 min coronary occlusion, each interspersed by 5 min reperfusions, before a subsequent 40 min ligation. To their surprise, the infarct size was reduced by almost 75% (5). These findings could not be explained by differences in collateral flow between preconditioned and control groups, and signalled the dawn of a new concept in myocardial cytoprotection. This original finding led to a very intense and extensive series of studies by various laboratories over the following decade to define cardiac adaptation to ischemia more precisely. Many models, species and various end points have thus far been investigated with conflicting, yet promising, results. The observed immediate myocardial adaptation is now termed ‘classical preconditioning’ or ‘early adaptation’ and is believed to last for 1 to 2 h following the preconditioning stimuli. Although there are various hypotheses as to the pathomechanism of preconditioning (7–9), this review focuses on the perceived role of ROS in the induction of cardiac adaptation to ischemia.
The first study to suggest a possible role for ROS in classical preconditioning was done by Murry and colleagues (10). They based their assumption on the observation that short ischemia-reperfusion cycles are bound to generate amounts of ROS that, although not detrimental, would be adequate to modify cellular functions, leading to a preconditioned state. They tested their hypothesis by administering superoxide dismutase (SOD) or catalase, both potent scavengers, during the preconditioning stimulus and partially abolished the previously afforded protection. Other researchers using various end points of cardioprotection have further confirmed these results. In a study by Osada et al (11) the antiarrhythmic effect of preconditioning was prevented by administration of SOD during the preconditioning stimulus. Administration of either SOD or N-2-mercaptopropionylglycine (MPG) was also shown to prevent protection against infarction in rabbit heart (12). Furthermore, in the absence of ischemia, the protective effect of preconditioning on infarct size and left ventricular function was mimicked in an isolated rabbit heart model subjected to low doses of oxygen radicals (13).
Nonetheless, in contrast to the positive studies cited, there were also numerous negative studies over the same period. Iwamoto et al (14) conducted an extensive study in which administration of SOD alone or in combination with catalase did not abolish the infarct limitation afforded by ischemic preconditioning, leading them to conclude that ROS are not involved in the mechanism of adaptation. More negative studies using other models have added fuel to the controversy (15,16). However, it can be argued that the discrepancy in observations can be attributed to the models or species employed. For instance, despite the similarity between the studies of Iwamoto et al (14) and Tanaka et al (12), one fundamental difference was in the number of cycles used to induce preconditioning. The former group used four cycles of ischemic preconditioning while the latter used only one, which may explain the differences in results. It is argued that with a more rigorous preconditioning stimulus (ie, multiple cycles) other triggers may be released in sufficient quantities such that eliminating one mediator would not adversely affect the induction of cardiac adaptation. This argument is supported by a study of in situ and in vitro rabbit hearts by Baines et al (17).
The complexity of the cellular cascade leading to a preconditioned state gives ground to the argument that ROS is a possible mediator of this adaptive mechanism (Figure 1). One step in this mechanism, recognized for its importance, is the activation of protein kinase C (PKC). A large body of evidence points to PKC activation as an integral step in the induction of preconditioning (18–20). Indeed, inhibition of PKC has been shown to abolish ischemic preconditioning in both rabbit hearts and isolated cardiomyocytes (18,21). Yet previously published reports had already defined a link between oxidative stress and PKC activation. Gopalakrishna et al (22) had shown that slight oxidative modification of the regulatory domain of PKC predisposes the enzyme to being more easily activated. These observations were further supported by the report that on exposure to oxidative stress, PKC is translocated from the cytoplasm to the cell membrane, where the enzyme is believed to exert its activity (23). A study by Tritto et al (13) also showed that PKC inhibition attenuates infarct reduction in a model of oxygen radical-induced preconditioning. These data suggest that ROS may mediate preconditioning through the activation of PKC. Another possibility is the reported ATP-dependent K+ (KATP) channels. These channels are assumed to have a major role in the pathogenesis of early cardiac adaptation to ischemia (24,25). Indeed, it has been shown that inhibiting these channels, for example by using glibenclamide, a KATP channel blocker, reverses the beneficial effects of preconditioning (26,27). Earlier observations in patch-clamp myocytes have shown that KATP channels are opened by various species of oxygen radicals. It is noteworthy that these results have also been confirmed in other cell types (28–30). The assumption that ROS can open KATP channels was later confirmed by results of a study by Pathak et al (31), in which the preconditioning effect of hydrogen peroxide was abolished by glibenclamide. Although many studies on classical preconditioning have hypothesized that the KATP channel is the likely end-effector of protection (27,32), very recently new evidence emerged to suggest otherwise (33,34). A study by Pain et al (33), investigating the critical time of KATP channel opening, concluded that contrary to previous data, these channels might be regarded as integral to triggering preconditioning. Furthermore, they suggested that opening of KATP channels before ischemia generates free radicals that lead to a preconditioned state and activation of kinases (33). Without elaborating further on other possible mechanisms, and considering the evidence available, it is reasonable to argue that ROS are key mediators of classical preconditioning.
In 1993 two independent studies by Marber et al (35) and Kuzuya et al (36) both showed that in addition to the initial phase (classical preconditioning), a second wave of protection appears almost 24 h following the preconditioning stimulus. Using rabbit and dog models, respectively, they observed that a 4×5 min preconditioning stimulus protected the heart against prolonged ischemia 24 h later. This delayed phase of protection is now referred to as the ‘second window of protection’ (SWOP), delayed preconditioning’ or ‘late preconditioning’, with certain characteristics distinct from classical preconditioning. It appears gradually, yet lasts as long as 72 h or more following the preconditioning stimulus. Although the protection it provides is not as pronounced as that afforded by classical preconditioning, it is postulated to be conveyed by newly synthesized cardioprotective proteins. They include heat shock proteins, the antioxidant Mn-SOD and nitric oxide synthase. These have been shown to increase in concentration and to be associated with infarct reduction in various studies of delayed preconditioning (37–40).
Potential roles for adenosine and nitric oxide in triggering delayed cardioprotection have been extensively investigated (37,41), and they have been linked to downstream activation of PKC and protein tyrosine kinase (PTK), both of which are thought to mediate delayed ischemic preconditioning (42–45). Indeed, inhibition of PKC abolished the cardioprotection afforded by SWOP in a number of models (42). The end-effectors of cardiac adaptation may differ between classic and delayed preconditioning, yet it is now generally accepted that their pathogenesis shares many common features. Although investigations of free radical signalling mechanisms in SWOP have used a limited range of species, plausible evidence points to an active involvement of these molecules. In the rat heart, Yamashita et al (38) proved that ROS were involved in delayed protection against infarction. In rat myocytes it has also been shown that bursts of ROS generated during the initial brief anoxic-reoxygenation periods lead to protection against prolonged anoxia and reoxygenation 24 h later (46). This effect was reproduced by exposure of the cells to exogenous superoxide anion. Delayed protection was defined as decreased lactate dehydrogenase release, reduced malondialdehyde formation, high energy phosphate preservation, improved cell morphology and viability in preconditioned and superoxide anion-treated myocytes, and increased Mn-SOD activity. In a conscious pig model, delayed protection against myocardial stunning was investigated by preconditioning with 10×2 min coronary occlusions (47). This protection was completely abolished if pigs were pretreated with a cocktail of antioxidants (SOD, catalase and MPG) before the preconditioning stimulus, thus concluding that ROS play an essential part in the genesis of delayed preconditioning. More convincing evidence has been provided by the comprehensive work of Das and colleagues (48), who provided direct evidence that ROS function as second messengers during cardiac adaptation.
Our own laboratory has also conducted a series of studies in an in vivo dog model, investigating ROS as triggers of delayed protection against infarction (49). Day 1 consisted of sham-operated or preconditioning protocols. Animals underwent 60 min of regional ischemia and 180 min of reperfusion 24 h later. Phase A of the study examined four groups in which infarct size in controls was compared with that in animals undergoing four, two or one cycle of preconditioning (5 min ischemia, 10 min reperfusion cycles). We observed significant protection in 4×5 and 2×5 preconditioned groups in comparison with the sham-operated group. The 1×5 stimulus led to a moderate but insignificant limitation in infarct size. In phase B of the study, MPG (antioxidant) was administered before preconditioning with similar protocols as in phase A. In the presence of MPG, the previous 2×5 and 1×5 preconditioning protections were abolished, yet significant protection was still seen with 4×5 preconditioning. These results led us to conclude that ROS contribute to triggering SWOP; however, this role seems to be overwhelmed when rigorous multiple cycles of ischemic preconditioning are employed. We hypothesize that with multiple cycles other triggers (adenosine, nitric oxide, etc) are perhaps released in sufficient quantity to induce cardioprotection, so that eliminating a single mediator (in this case ROS) would not have diminished the overall stimulation to a subthreshold level. This assumption closely resembles the observations by Iwamoto et al (14) and Tanaka et al (12) in studies on ROS in classical preconditioning discussed previously.
PKC activation and translocation have been shown as integral in the intracellular signalling processes leading to delayed cardiac adaptation (42,50). It is suggested that PKC can activate gene transcription, yielding proteins instrumental in delayed cardioprotection (51). One hypothesis is that ROS can lead to delayed adaptation through a PKC-dependent pathway (Figure 1). ROS, nitric oxide and PKC are intricately linked in the induction of SWOP in the nitric oxide hypothesis proposed by Bolli et al (37). They suggested that the abundant ROS and nitric oxide generated during brief ischemic episodes may either act independently or react with one another to form other radicals capable of activating PKC. Activated PKC can then lead to transcriptional activation of inducible nitric oxide synthase, which is responsible for generation of nitric oxide, argued to confer delayed cardioprotection.
More recently, evidence has been emerging on the induction by ROS of a PTK-dependent pathway leading to cardio-protection. Maulik and colleagues (52) showed that ROS provoke a signal transduction pathway involving PTK coupled with phospholipase D that functions either independently or downstream of PKC, leading to the activation of other kinases. A study of delayed protection against myocardial stunning also defined a bifunctional role for PTK, necessary in induction of preconditioning, as well as mediating protection 24 h later (45). Downstream, the PTK-dependent signal trasduction leads to enhanced phosphorylation and activation of mitogen-activated protein kinase (such as p38 MAP kinases), which ultimately targets other kinases that may lead to phosphorylation of smaller heat shock proteins induced by ischemic preconditioning (48,52,53). The nuclear transcription factor NFκB has also been recognized to play a crucial part in delayed preconditioning. Maulik and colleagues (54) showed that, in the setting of ischemic preconditioning, activation of the MAP kinase cascade leads to activation of NFκB, which in turn induces gene expression associated with cardioprotection. There is also strong evidence that oxidative stress can lead to NFκB translocation and activation (54–56). Thus, overwhelming data support the possible role of ROS in triggering the cellular cascades that lead to delayed ischemic adaptation, through either a PKC-dependent or a PKC-independent pathway.
The cellular signalling cascades leading to ischemic preconditioning have been exhaustively investigated. Although much has been learned about the pathomechanism of both classical and delayed cardiac adaptation to ischemia, the only certainty is that the genesis of preconditioning is a very complex and intricate process. Many factors can work independently or in concert, sharing many common pathways. Whatever the downstream mechanism, ROS have been documented to play an active part in the induction of both immediate and delayed cardioprotection. This concept brings into question the attitude adapted wholesale toward benefits of antioxidant therapy. Although the toxicity of ROS has been conclusively outlined, it is arguable that, under milder conditions, oxygen radicals can perhaps double as a warning bell, ultimately leading to increased cell tolerance and performance, as is the case with cardiomyocytes. Hence, although we do not question the validity or justification of antioxidant therapy, the question arises as to under what conditions and timeframes one should embark on this form of intervention, bearing in mind the benefits of exposure to a mild oxidative stress. To that end more investigations are deemed necessary to define clearly the dimensions and boundaries of advantageous oxidative modulation of cellular functions, against the detrimental and sometimes irreversible injuries of oxidative stress.
This work was supported by Hungarian Research Fund ETT 369-02.