Tao Rui Gediminas Cepinskas and Peter R Kvietys

Vascular Biology/Inflammation Program, Lawson Health Research Institute, London, Ontario, Canada

Introduction

Reperfusion of ischemic myocardium results in pathological changes in the heart, such as myocardial stunning, an acute inflammatory response, and infarction. Paradoxically, pretreatment of the heart with an ischemia-reperfusion (I/R) challenge confers protection against a subsequent I/R insult. This phenomenon, referred to as ischemic preconditioning (PC), has two distinct phases based on the time course and mechanisms involved. An early phase of PC occurs within minutes after the initial I/R challenge and persists for only a few hours. This early PC is independent of protein synthesis and, thus, relies on activation of existing effector molecules. There is also a later phase of PC, which becomes apparent 24 hours or later after the initial I/R challenge and persists for up to several days (delayed PC). Delayed PC is dependent on protein synthesis and, thus, represents a genetically mediated adaptive response. Herein, we will focus on some of the proposed mechanisms involved in the development of delayed PC with respect to the initiating (reactive oxygen metabolites), signaling (nuclear transcription factors), and effector (nitric oxide synthase and superoxide dismutase) components.

The heart is a complex organ consisting of different cell types with specialized functions (myocytes, endothelial cells, and so on). These individual cells may respond differ ently to I/R and use distinctly different mechanisms to contribute to delayed PC in the myocardium. Thus, both in vivo and in vitro approaches have been used to address the mechanisms involved in the development of delayed PC. In vivo studies generally involve an initial series of short I/R challenges to the heart followed 24 hours (or later) by a more prolonged I/R challenge (Figure 1A). The in vitro approaches simulate I/R by targeting the oxygenation aspects of I/R. Typically isolated cardiac myocytes are exposed to anoxia (or hypoxia) and, subsequently, reoxy-genated (anoxia/reoxygenation; A/R) (Figure 1B).

Initiating Molecules

Reactive oxygen metabolites (ROM) are generated within cells as a normal consequence of aerobic metabolism. Mammalian cells contain antioxidant enzymes (cata-lase, SOD, and so on) to scavenge and neutralize these oxidants. It is generally believed that the oxidant production after an I/R challenge overwhelms the detoxifying capacity of the endogenous antioxidants and initiates tissue injury.

The following lines of evidence support a role for ROM in the initiation of delayed PC in vivo. An I/R challenge to the heart in situ results in the local generation of oxidants. Administration of antioxidants during the initial I/R

Figure 1 Current hypotheses based on in vivo (A) and in vitro (B) studies on the mechanisms involved in the development of delayed preconditioning. (A) In vivo studies involve an initial series of short ischemia-reperfusion (I/R) challenges (e.g., six cycles of 4-minute coronary artery occlusion followed by 4-minute reperfusion) and 24 hours later challenged with a more prolonged period of I/R (e.g., 30-minute period of occlusion and one 24-hour period of reperfusion). (B) In vitro studies involve exposing isolated cells to a 30-minute period of anoxia, reoxygenating them (A/R), and 24 hours later subjecting these cells to the same A/R protocol. See text for the lines of evidence supporting the proposed initiating (e.g., oxidants), signaling (nuclear transcription factors), and effector (NOS and SOD) components of the development of delayed preconditioning.

Î Cytotoxicity Cytotoxicity

PMN Emigration t PMN Emigration

Figure 1 Current hypotheses based on in vivo (A) and in vitro (B) studies on the mechanisms involved in the development of delayed preconditioning. (A) In vivo studies involve an initial series of short ischemia-reperfusion (I/R) challenges (e.g., six cycles of 4-minute coronary artery occlusion followed by 4-minute reperfusion) and 24 hours later challenged with a more prolonged period of I/R (e.g., 30-minute period of occlusion and one 24-hour period of reperfusion). (B) In vitro studies involve exposing isolated cells to a 30-minute period of anoxia, reoxygenating them (A/R), and 24 hours later subjecting these cells to the same A/R protocol. See text for the lines of evidence supporting the proposed initiating (e.g., oxidants), signaling (nuclear transcription factors), and effector (NOS and SOD) components of the development of delayed preconditioning.

challenge prevents the development of delayed preconditioning with respect to myocardial stunning and infarct size. Local administration of oxidant-generating chemicals (rather than an initial I/R challenge) can induce the development of delayed PC.

In vitro studies indicate that an oxidant stress is also important in initiating the development of delayed PC in isolated cardiac myocytes. Exposure of myocytes to a mild A/R challenge (30/30 minutes) induces (1) an oxidant stress and (2) the rapid development of a proinflammatory phenotype (these myocytes can promote PMN transendothelial migration). Interestingly, the in vitro studies have uncovered an important communication link between myocytes and endothelial cells, that is, the A/R-induced increase in myocardial oxidant stress is transferred to the adjacent endothelial cells and contributes to the PMN transendothelial migration [1]. When cardiac endothelial cells derived from mice overexpressing Mn-SOD are used in assays, the A/R-challenged myocytes no longer induce an oxidant stress in the adjacent endothelial cells and the myocytes no longer promote PMN transendothelial migration (Figure 2). A more severe A/R challenge (1 hour of anoxia) can also induce cell death in some of the myocytes. If the myocytes are pretreated with a mild A/R challenge 24 hours earlier, (1) the subsequent A/R challenge does not result in an oxidant stress, (2) a proinflammatory phenotype does not develop, and (3) there is less cytotoxicity (delayed PC). Delayed PC can also be induced by pretreatment of the myocytes with H2O2 rather than an A/R challenge. Finally, delayed PC can be prevented by pretreating the myocytes during the initial A/R challenge with an antioxidant.

Taken together, both in vivo and in vitro studies indicate that induction of an oxidant stress during the initial I/R or A/R challenge plays an important role in initiating the development of delayed PC (Figure 1).

The source of the ROM within the myocytes is not clear and appears to be controversial. There are three isoforms of NOS in the myocardium: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Of these three isoforms, eNOS appears to be involved in the initiation

Figure 2 Endothelial cell oxidant stress (top) and PMN transendothelial migration (bottom) induced by supernatants from A/R-conditioned myocytes was diminished when endothelial cells derived from hearts of Mn-SOD overexpressing mice were used. From Rui et al. (2001). Am. J. Physiol. Heart Circ. Physiol. 281, H440-H447.

Figure 2 Endothelial cell oxidant stress (top) and PMN transendothelial migration (bottom) induced by supernatants from A/R-conditioned myocytes was diminished when endothelial cells derived from hearts of Mn-SOD overexpressing mice were used. From Rui et al. (2001). Am. J. Physiol. Heart Circ. Physiol. 281, H440-H447.

of delayed PC in the heart. Brief periods of I/R challenge are associated with NO biosynthesis, the most likely source being eNOS. Based on pharmacological approaches, NO generated by eNOS appears to play a role in initiating delayed PC in the heart with respect to stunning and infarction. Administration of nonspecific NOS inhibitors (but not specific iNOS inhibitors) prior to the initial I/R challenge prevents delayed PC. Conversely, local administration of NO donors (rather than an initial I/R challenge) can induce delayed PC. Taken together, these observations suggest that NO derived from eNOS initiates the development of delayed PC (presumably via the generation of ROM). In vitro studies using isolated cardiac myocytes are not in agreement with the in vivo studies. The initial A/R challenge does not result in the production of NO, and pharmacological blockade of NO synthase does not prevent the oxidant stress induced by the initial A/R challenge. Furthermore, the initial A/R challenge can still induce an oxidant stress in cardiac myocytes isolated from eNOS deficient mice. Similar results have been noted with cardiac endothelial cells, that is, the initial A/R challenge induces an oxidant stress in endothelial cells harvested from eNOS-deficient mice.

Signaling Molecules

Of the potential nuclear transcription factors that could be involved in the development of delayed PC, NFkB has received the most attention. ROM can activate NFkB, thereby allowing it to translocate to the nucleus. Increased levels of NFkB have been noted in nuclear extracts obtained from hearts subjected to I/R, an effect prevented by the administration of an antioxidant during the I/R challenge. Furthermore, pharmacological prevention of NFkB activation and translocation to the nucleus abrogates the development of delayed PC with respect to myocardial stunning and infarction. In vitro studies are in general agreement, that is. an initial A/R challenge imposed on cardiac myocytes results in the mobilization of NFkB to the nucleus. A similar observation was noted using isolated human umbilical vein endothelial cells (HUVECs). Pharmacological prevention of NFkB activation or translocation to the nucleus after the initial A/R challenge to cardiac myocytes prevented the development of delayed PC with respect to (1) an oxidant stress and (2) the development of a proinflammatory pheno-type. That is, under these conditions, the oxidant stress and myocyte-mediated PMN transendothelial migration was the same after the initial and second A/R challenges. In HUVECs, introduction of an oligonucleotide with consensus binding sites for NFkB during the first A/R challenge prevented the development of delayed PC with respect to PMN adhesion to HUVECs. Thus, both in vivo studies and in vitro studies support a role for NFkB as an important signaling transcription factor in the development of delayed PC (Figure 1).

In vitro studies have also uncovered a potential role for another nuclear transcription factor in delayed PC in cardiac myocytes. After the initial challenge there was an increased accumulation of AP-1 in the nucleus. Furthermore, introduction of an oligonucleotide containing binding sites for AP-1 during the first A/R challenge prevented the development of delayed PC with respect to (1) myocyte oxidative stress and (2) myocyte-mediated PMN transendothelial migration (unpublished observation).

Effector Molecules

Superoxide Dismutase (SOD)

Administration of antioxidant enzymes (e.g., SOD, cata-lase) can ameliorate I/R-induced myocardial injury. Thus, it has been proposed that an increase in the antioxidant status of the heart can contribute to the development of delayed PC. Of the endogenous antioxidants that are upregulated after an I/R challenge, SOD (particularly Mn-SOD) has received the most attention. Overexpression of Mn-SOD (transgenic mice) results in a reduction in the I/R-induced myocardial infarct size. Furthermore, endogenous Mn-SOD protein and activity are increased in the myocardium during the development of delayed PC. There appears to be a direct correlation between the increase in myocardial Mn-SOD activity and the reduction in infarct size. Finally, inhibition of Mn-SOD protein synthesis and activity by the use of an antisense approach prevents the development of delayed PC with respect to infarct size. In isolated cardiac myocytes, Mn-SOD, but not Cu/Zn-SOD, activity is increased 24 hours after the initial A/R challenge. Pretreatment of the myocytes with a Mn-SOD antisense oligonucleotide prior to the initial A/R challenge (to prevent Mn-SOD synthesis) prevents the increase in SOD activity typically observed 24 hours after the initial A/R challenge. In addition, this procedure also prevents the development of delayed PC with respect to (1) PMN transendothelial migration and (2) myocyte death. Collectively, these in vivo and in vitro observations provide strong support for the contention that the development of delayed PC is dependent on the induction of Mn-SOD (Figure 1).

The in vitro studies have also provided some strong evidence favoring a role for NFkB as the nuclear transcription factor involved in upregulating Mn-SOD. Pharmacological inhibition of NFkB activation or translocation during the initial A/R challenge prevents the induction of Mn-SOD. In addition, this maneuver prevents the development of delayed PC with respect to (1) myocyte oxidative stress and (2) myocyte-mediated PMN transendothelial migration.

Nitric Oxide Synthase (NOS)

Of the three isoforms of NOS (eNOS, nNOS, and iNOS), eNOS and iNOS appear to be involved in the development of delayed PC in the heart.

The iNOS isoform appears to play an important role as an effector molecule in the development of delayed PC at the whole organ level (Figure 1A). NO production is increased in the myocardium at 24 hours after the initial I/R challenge. Message levels of iNOS are increased within 3 hours after the initial I/R challenge, and iNOS protein and activity are increased at 24 hours. By contrast, eNOS protein and activity are unaltered at 24 hours after the initial I/R insult. Administration of selective iNOS inhibitors during the second I/R challenge prevents delayed PC with respect to both myocardial stunning and infarction. Finally, delayed PC (infarct size) is abrogated in iNOS-deficient mice [2].

In vitro studies have yielded strikingly different conclusions regarding the role of eNOS and iNOS as effector enzymes in the development of delayed PC [3]. The initial challenge of myocytes derived from iNOS-deficient mice resulted in an increase in myocyte oxidative stress. If these myocytes were pretreated with an A/R challenge 24 hours earlier, the second A/R challenge did not induce an increase in myocyte oxidant stress (delayed PC). In a similar fashion, the initial challenge of myocytes derived from eNOS-deficient animals resulted in an increase in myocyte oxidant stress. However, an A/R challenge imposed 24 hours after an initial A/R challenge to myocytes from eNOS-deficient mice still resulted in an increase in myocyte oxi-dant stress (no delayed PC). Identical results were obtained using isolated cardiac endothelial cells, rather than myocytes in the in vitro assays, that is, delayed PC (with respect to an oxidant stress) could be demonstrated in endothelial cells derived from iNOS-deficient mice, but not in those derived from eNOS-deficient mice. In myocytes derived from wild-type mice, mRNA for eNOS, but not iNOS, increased after the initial A/R challenge. Similarly, mRNA for eNOS increased after an A/R challenge imposed on myocytes derived from iNOS-deficient mice. Taken together these findings indicate that eNOS-derived NO is a prerequisite for the development of delayed PC in isolated cardiac myocytes (Figure 1B).

The reason for the disparate results between in vivo and in vitro studies regarding the isoform of NOS (iNOS versus eNOS) serving as an effector enzyme in delayed PC is not entirely clear. However, one possible explanation deserves comment. The in vivo studies targeted the heart in situ with an intact systemic circulation, while the in vitro studies used cardiac myocytes isolated from their natural external milieu. Based on studies of the systemic inflammatory response syndrome, it is quite possible that multiple I/R challenges to the heart in vivo would result in the systemic accumulation of cytokines, which in turn would impinge upon the heart during the 24-hour hiatus before the second I/R challenge. Although I/R, per se, is a weak stimulus for induction of iNOS, cytokines are potent stimuli for induction of iNOS. Thus, in the in vivo studies it is quite possible that cytokines contributed to the I/R-induced iNOS upregulation. Such an effect would be precluded in the in vitro studies. Further, studies are warranted to directly assess this possibility.

The in vitro studies have also provided evidence supporting a role for AP-1 in upregulating eNOS protein and activity. Pretreatment of cardiac myocytes with an oligonu cleotide that contains binding sites for AP-1 (prevention of AP-1 translocation to the nucleus) during the initial A/R challenge abrogates the A/R-induced upregulation of eNOS. In addition, this maneuver prevents the development of delayed PC with respect to (1) myocyte oxidative stress and (2) myocyte-induced PMN transendothelial migration (unpublished observation).

Interaction between NOS and SOD

The in vitro studies indicate that both eNOS and Mn-SOD appear to be critical effector enzymes in the development of delayed PC. These two enzyme systems may be acting in concert. In myocytes isolated from either iNOS-deficient mice (which developed delayed PC) or eNOS-deficient mice (which did not develop delayed PC), Mn-SOD protein increased to the same extent 24 hours after the initial A/R insult. However, in myocytes isolated from iNOS-deficient mice Mn-SOD activity was increased, whereas in myocytes derived from eNOS-deficient mice Mn-SOD activity was not affected. Collectively, these observations indicate that NO derived from eNOS is modulating the activity of Mn-SOD, but not affecting its transcription [3]. The exact mechanism(s) by which eNOS-derived NO is modulating Mn-SOD activity under these conditions is not entirely clear and warrants further attention.

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