Cardiovascular Research

Cardiovascular Research 1998 40(1):9-22; doi:10.1016/S0008-6363(98)00142-4
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Role of protein kinase C as a cellular mediator of ischemic preconditioning: a critical review

Boris Z. Simkhovich^*, Karin Przyklenk and Robert A. Kloner

Heart Institute, Good Samaritan Hospital and Department of Medicine (Section of Cardiology), University of Southern California, Los Angeles, CA 90017, USA

^* Corresponding author. Tel.: +1 (213) 977 4194; Fax: +1 (213) 977 4107.

Received 15 January 1998; accepted 22 January 1998

	Abstract

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
Protein kinase C (PKC) has been proposed as a primary cellular mediator of ischemic preconditioning. However, the role of PKC in eliciting cardioprotection remains controversial. In this review, we summarize the evidence for and against the ‘PKC hypothesis’ of preconditioning, discuss the important technical limitations currently hampering PKC research, and suggest new approaches (i.e. PKC isoform-specific biochemical assays combined with immunoblotting techniques) that might aid in the definite resolution of this issue.

KEYWORDS Ischemic preconditioning; Protein kinase C; Signal transduction

	1 Introduction

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
Ischemic preconditioning is a phenomenon of endogenous protection that renders the heart resistant to sustained ischemia [1–7]. It is generally accepted that this protection is a receptor-mediated process, and is realized via signal transduction pathways.

A signaling factor proposed to be integral in ischemic preconditioning is protein kinase C (PKC) [8–13]. Our objective in this review is to summarize the role of PKC in the general mechanism of cellular processes in the heart, discuss the evidence for and against PKC as a mediator of the cardioprotection achieved with brief preconditioning ischemia, and provide suggestions as to how the controversies regarding the role of PKC in preconditioning might be resolved.

	2 The function of PKC in the heart

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
PKC catalyzes the transfer of -terminal phosphate from ATP to the hydroxyl group of serine and/or threonine (Ser/Thr) residues in various protein substrates, and this process is considered to be a fundamental regulatory mechanism involved in cellular growth, differentiation and immediate regulation of effector functions [14–17]. One of the most important roles of PKC is the activation of transcription. During PKC-mediated activation of transcription, constituents of the immediate early gene program expression (i.e. c-fos and c-jun) form a heterodimeric activator protein-1 complex (AP-1) which binds to the TRE sequence (12-O-tetradecanoylphorbol β-acetate responsive element) within the promoter region. It is this binding to the responsive element that activates gene expression [16]. In cardiomyocytes, direct involvement of PKC in this process was confirmed for genes expressing light chain 2 of cardiac myosin, atrial natriuretic factor, and the cardiac isoform of sarcoplasmic Ca²⁺-ATPase [16, 18–20]. PKC-mediated gene activation also was suggested to be involved in cardiac hypertrophy [17].

PKC has further been implicated in immediate functional regulation of cardiac proteins via their direct phosphorylation. For example, PKC has been reported to catalyze phosphorylation of troponin I and troponin T subunits in bovine cardiac troponin complex [21]. Since this study was conducted by employing isolated cardiac proteins, it is not clear whether these in vitro findings are physiologically relevant. PKC has been identified as a regulator of heart contractility, yet studies with phorbol esters (positive modulators of PKC activity) have been equivocal. There are reports indicating that phorbol ester can increase the contractility of isolated adult rat ventricular myocytes [22], while other authors, utilizing rat ventricular myocytes and isolated perfused rat hearts, demonstrated a negative inotropic effect, the latter being concentration- and time-dependent [23–25].

Several recent studies indicate that PKC can modulate ion currents in cardiomyocytes. PKC activates cardiac ATP-sensitive K⁺ channels, and thus acts as a K_ATP channel opener [26, 27]. Increased PKC association with ventricular cardiomyocyte membranes also resulted in a decreased Ca_i transient and thus might explain the negative inotropy induced in some studies by phorbol esters [23].

Despite ongoing controversies, these studies clearly reveal that PKC can be involved in several important mechanisms underlying different types of cellular effects. PKC-mediated effects on contractile proteins and ion channels may underlie immediate responses to physiological and/or pathological stimuli, while participation in the regulation of the genome may indicate its involvement in such fundamental cellular processes as growth and differentiation.

	3 Cardiac PKC isoforms in different species

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
The PKC family consists of 12 closely related Ser/Tre kinases, classified into three distinctive subfamilies. The subfamily of conventional, or classical PKC isoforms, includes PKC , β₁, β₂ and isoforms, and requires both calcium ions and lipids (i.e. phosphatidylserine, PMA and/or diacylglycerol) for their activation. The subfamily of novel PKC isoforms, which includes PKC , , , and µ, does not require calcium, but, as with the classical isoforms, still requires lipids for their activation. The subfamily of atypical PKC isoforms includes PKC , , and . PKC requires phosphatidylserine for its activity, but neither calcium ions, nor diacylglycerol and/or PMA are needed for its activation.

In adult rat hearts, novel PKC and are the major isoforms. Minor PKC isoforms are represented by novel PKC and atypical PKC and [11, 28–30]. Conventional calcium-dependent PKC isoforms represent only a very small portion of all PKC expressed in the adult rat heart, and are believed to originate from cellular elements other than myocytes [29]. In adult rabbit hearts, immunoblotting revealed the presence of conventional PKC, PKCβ₁, PKCβ₂ and PKC, novel PKC and atypical PKC [31]. In a recently published study 10 individual PKC isoforms were found in rabbit heart; i.e. PKC , β₁, β₂, , , , , , , and µ. The authors also demonstrated that conventional PKC isoforms (i.e. , β₁, β₂, and ) are more abundant than the novel PKC isoforms [13]. In contrast, in adult dog hearts, the two major PKC isoforms are and [17], while in human cardiac tissue, PKC is the major isoform, and the minor isoforms are represented by PKC , , , and [30].

The biological significance of this marked PKC isoform diversity among species has not been clarified fully. Only a few studies are available indicating isoform selectivity in terms of regulating gene expression and substrate modification. In this regard Kariya et al. reported that PKCβ preferentially activated the transcription of β-myosin heavy chain, while PKC was only a modest activator of its transcription [32]. Isoform-specific substrate modification was also confirmed for troponin I and troponin T from bovine troponin complex. PKC caused a decrease in Ca²⁺ sensitivity of actomyosin Mg-ATPase by phosphorylating Ser-23/Ser-24 residues in troponin I molecules. PKC, on the other hand, was shown to be unique in its selective phosphorylation of specific sites in the troponin T molecule and in causing an increase in actomyosin Ca²⁺ sensitivity [21].

	4 The concept of PKC translocation

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
Under baseline conditions, PKC is rendered largely inactive by intramolecular interaction between ‘pseudosusbstrate’ and catalytic sites. Pseudosubstrate represents a part of the structure of PKC that interacts with its active site and causes PKC autoinhibition. PKC activation weakens conformational interactions between the pseudosubstrate and the catalytic sites, and makes the pseudosusbstrate sequences less accessible to the PKC catalytic moiety [33].

Calcium-dependent PKC isoforms from various tissues have been shown to be stimulated by diacylglycerol, in the presence of calcium ions and phospholipids (e.g. phosphatidylinositol, phosphatidylserine and phosphatidic acid). Diacylglycerol, containing unsaturated fatty acids, was identified as the most active constituent, capable of enhancing the phospholipid-dependent PKC activation [34, 35]. By 1980 it was known that phosphatidylinositol is one of the most rapidly metabolized cellular phospholipids in response to various extracellular messengers, is mainly composed of unsaturated fatty acids, and diacylglycerol is one of its first turnover products. Based on these facts it was suggested that phospholipase C-mediated cleavage of phosphatidylinositol may be crucial for PKC activation [35]. Tumor-promoting phorbol ester (i.e. 12-O-tetradecanoylphorbol 13-acetate, TPA) was also able to activate Ca²⁺-dependent PKC directly and to increase PKC affinity for calcium ions significantly [36]. In parietal yolk sac (PYS) cells TPA was able to increase dramatically the association of PKC with the plasma membrane, accompanied by its decrease in the cytosol [37]. Similarly, diacylglycerol also was shown to promote PKC association with the membrane fraction of PYS and NIH 3T3 cells [38]. More recent studies have reported that diacylglycerol significantly increases PKC binding to phosphatidylcholine/phosphatidylserine-containing membranes with high selectivity towards the phosphatidyl-L-serine moiety [39, 40]. A cysteine-rich region within PKC regulatory domain was implicated in the diacylglycerol- and phorbol ester-dependent regulation of PKC activity and its subcellular distribution [41–43]. As a result of all the above-mentioned studies, the concept of PKC translocation was formulated.

PKC activation and subsequent translocation to membrane compartments is a key event in the PKC-dependent signaling cascade, and is preceded by agonist-mediated stimulation of receptor-associated phospholipase C (PLC). PLC, upon stimulation, cleaves inositol-1,4,5-trisphosphate and diacylglycerol from the membrane-bound phosphatydylinositol moiety. Diacylglycerol acts as a hydrophobic factor, and increases the affinity of PKC to the membrane-integrated structures within the subcellular compartments [15, 44]. In other words, diacylglycerol facilitates the translocation of PKC into these compartments.

Until recently, it was generally accepted that PKC, upon its translocation, binds to lipid moieties within the membrane compartment. However, immunological and biochemical studies suggest that PKC may become attached to specific proteins within the membrane and nuclear subcellular fractions. Intracellular receptors for activated PKC (RACK proteins; i.e. receptors for activated C-kinase) have been described, and some of them were successfully cloned [45–47]. Their ability to interact specifically with PKC molecules was tested in experiments in which purified rat brain RACKs, injected into Xenopus oocytes, competed for PKC with endogenous RACKs and inhibited the maturation of oocytes [48]. The discovery of PKC-anchoring proteins is a significant step toward our understanding of the complexity of the PKC-mediated signaling cascade, and may provide an explanation of how different PKC isoforms are translocated to different subcellular compartments.

PKC translocation has been documented for many conventional, novel and atypical PKC isoforms in the heart, but its biological significance is still far from being resolved. The recent discovery of RACK proteins may provide additional support for the PKC translocation concept. These proteins may work in concert with the PKC activation process both by directing activated PKC molecules to specific subcellular compartments and stabilizing their active state within these particular compartments. However, despite the fact that the translocation of PKC is considered a key step in PKC-mediated signaling, PKC activation, not followed by its translocation, cannot be ruled out.

	5 Measurement of PKC activity and its subcellular distribution

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
The unusual properties of PKC (i.e. its translocation to different subcellular compartments upon activation) complicates the quantitative assessment of this enzyme. For example, the assay cannot be performed in whole tissue homogenates; rather, subcellular fractionation is mandatory. During subcellular fractionation, possible cross-contamination between subcellular compartments deserves serious consideration. In this regard, a detailed characterization of isolated fractions by electron microscopy and/or measurement of marker enzymes is necessary. Finally, the fact that there are 12 individual PKC isoforms may result in the requirement of performing isoform-specific measurement of PKC activity.

Most of the radioactive PKC assay methods are based on the incubation of isolated subcellular fractions with -³²P-ATP in the presence of PKC specific substrate. The reaction product (i.e. phosphorylated substrate) is isolated on phosphocellulose filters or by employing Avidin–Biotin technology and counted in a β-scintillation counter (Fig. 1). In non-radioactive kits, phosphorylated peptide is detected using ELISA technique or other physical and/or chemical methods. Commercially available radioactive and nonradioactive PKC assay systems are summarized in Table 1.

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Subcellular distribution of protein kinase C (PKC) activity measured in rabbit heart using a radioisotope-based biochemical method (Amersham PKC assay kit). In the in vivo model of coronary artery occlusion (Panel A), PKC activity in the cytosolic (data not shown for simplicity) and particulate fraction (containing nuclei and membranes) was comparable in both nonischemic control hearts and hearts that received 2 5-min episodes of preconditioning (PC) ischemia. That is, there was no evidence of PKC translocation to the particulate fraction in response to brief, in vivo regional ischemia. Adapted from reference [56]. In contrast (Panel B), in the excised rabbit heart, PKC activity in the membrane and nuclear compartments was increased following only 2 min of normothermic global ischemia when compared with matched baseline samples. Adapted from reference [103].

 

View this table: [in this window] [in a new window]   Table 1 Protein Kinase assay kits

 
Unfortunately, all currently available PKC substrates demonstrate only limited specificity (Table 2). As a result, there is a danger that activation and/or translocation of a minor PKC isoform may not be detected. There are several peptides that can successfully distinguish activities of conventional plus novel PKC isoforms (i.e. EGF-derived peptide and MBP-derived peptide), and activities of PKC and (PKC pseudosusbstrate-derived peptide), but none of them allows the specific determination of activities of individual PKC isoforms present in a particular species, tissue and/or subcellular compartment. However, it may be possible to develop this kind of technique. Isoform-specific antibodies to all PKC isoforms detected in rat and rabbit hearts are currently available. Thus, by making corresponding combinations of PKC-specific antibodies and PKC substrates, it should be possible to prepare a reaction mixture which will detect only one PKC isoform, while all other isoforms present in the sample of interest will remain blocked by their antibodies. If developed, this method could become a very useful tool in PKC-related studies.

View this table: [in this window] [in a new window]   Table 2 PKC Substrates

 
PKC immunoblotting represents a valuable alternative to biochemical assessments, and has been successfully employed by numerous laboratories worldwide. This technique utilizes electrical current to isolate proteins present in processed tissue samples and/or subcellular fractions, and to transfer them (i.e. to blot) to nitrocellulose filter, or nylon membrane. Nonspecific sites of blotted proteins are blocked with milk proteins, and primary antibodies against individual PKC isoforms are added. After incubation and washing, all proteins blotted and tagged with primary antibody are incubated with secondary horseradish peroxidase-tagged antibody, allowing the investigator to perform qualitative and/or semiquantitative analysis of the immunological reaction between individual PKC isoforms and the primary antibody. The bound antibody can be detected by reading the luminescence produced after the oxidation of luminol by horseradish peroxidase (ECL Immunoassay Signal Reagent, Amersham, Arlington Heights, IL). This technique provides the investigator with information regarding the presence and location of individual PKC isoforms, but does not determine its activity status. Since the association with membranes does not necessarily reflect the state of activation, the lack of PKC activity data is an important limitation of immunoblotting technique [15].

The activity and subcellular distribution of PKC has also been studied in isolated cells and frozen tissue samples by labeling PKC with cell-permeable fluorescent probes (e.g. PKC inhibitor bisindolylmaleimide conjugated to fluorescein): [49, 50]. Although not isoform-specific, this approach has the unique advantage that the fluorescent probe binds to the catalytic domain of PKC and thus is selective for active enzyme [49, 51]. Since there are numerous PKC inhibitors with different isoform specificity, this method may represent an attractive alternative to the above-mentioned immunoblotting technique.

Another important issue is the cellular origin of PKC isoforms found in the heart: i.e., without knowing whether the PKC isoforms are present in myocytes, endothelial cells, etc., it is difficult to determine whether changes in activity and/or subcellular distribution are relevant to myocardial ischemia, and represent specific changes within the cardiomyocytes. For example, Ping and colleagues recently reported the presence of 10 individual PKC isoforms in adult rabbit heart [13]. However, without the information regarding their cellular location it is not clear whether changes in the subcellular distribution of PKC isoforms are taking place solely in cardiomyocytes.

Thus, in summary, we at present do not have a reliable and quantitative method capable of determining the activity and subcellular localization of specific isoforms of PKC. To address this problem, an isoform-specific immunoblotting technique needs to be combined with a biochemical isoform-specific assay. The latter can be developed by employing isoform-specific antibodies and already existing PKC activity assay methods. Combination of immunoblotting technique with this prospective biochemical method may overcome limitations of currently available PKC assay systems. Alternatively, Johnson and colleagues recently introduced a PKC-specific peptide and isoform-selective PKC translocation inhibitor into cultured neonatal cardiac cells [52, 53]. This technique lays the groundwork for selective labeling of myocardial and non-myocardial cells with PKC specific substrates and/or translocation inhibitors in vivo, by utilizing differences in surface receptors and/or other cell-specific markers.

	6 PKC and ischemic preconditioning

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
Despite almost ten years of intensive study, the cellular mechanism responsible for preconditioning still remains obscure. One of the most popular hypotheses implicates the translocation of PKC from the cytosol to the sarcolemma and subsequent phosphorylation of an as-yet unknown protein target as key events in achieving cardioprotection. A large body of experimental data, largely based on indirect pharmacological evidence, has supported the ‘PKC hypothesis’ of preconditioning [8, 9, 54]. However, direct measurements of the subcellular distribution of PKC have failed to conclusively demonstrate its involvement in the protective effect of ischemic preconditioning [49, 55, 56]. The role of PKC in preconditioning has been studied in rats, rabbits, dogs and swine, both in vivo and in vitro, utilizing different experimental models and preconditioning protocols. In this section, we review the current controversies regarding the involvement of PKC in acute protection elicited by intermittent brief episodes of ischemia and reperfusion (i.e., so-called ‘classical’ preconditioning).

	7 Studies in rabbits

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
The PKC hypothesis of ischemic preconditioning was first formulated by Downey and colleagues using the rabbit model. Specifically, Ytrehus et al. demonstrated that either staurosporine and polymyxin B, when given in vivo 5 min before a 30 min sustained occlusion (i.e. midway during the 10 min period of intervening reflow), were able to block the protective effect of ischemic preconditioning [8]. Later, Liu et al., from the same group, further postulated that PKC translocation during brief episodes of preconditioning ischemia underlies the protective effect of ischemic preconditioning. In their in situ studies they demonstrated that staurosporine, administered 5 min before the preconditioning episode, did not abolish the protective effect of ischemic preconditioning. However, when microtubules within the cardiomyocytes were purportedly disrupted with colchicine, the protective effect of ischemic preconditioning was blocked [9]. Based on their observations, the authors concluded that PKC activity is not required during the preconditioning episodes, and the integrity of the membrane compartment is crucial for the protection afforded by ischemic preconditioning. According to their hypothesis, preconditioning ischemia causes PKC translocation into the membrane compartment, facilitating phosphorylation of an as-yet unknown protein target during sustained ischemia. Importantly, however, neither PKC translocation, nor the disruption of microtubules by colchicine, were confirmed directly.

In a recently published paper Downey's group expanded upon their initial observation. Using isolated perfused rabbit hearts subjected to brief global preconditioning ischemia and sustained regional coronary occlusion, they reported that staurosporine blocked the protective effect of ischemic preconditioning only when it was present in the perfusion medium following the episode of preconditioning ischemia (i.e. during the intervening reperfusion) and during the early phase of sustained occlusion. In contrast, the protective effect of ischemic preconditioning was still present when the PKC inhibitor was added before preconditioning ischemia/reperfusion, but washed out before the sustained occlusion [54]. Based on their results the authors concluded that ischemic preconditioning is a two-phase phenomenon. The first phase serves as a trigger, does not require PKC-mediated phosphorylation, and only involves PKC translocation. This phase occurs during the preconditioning stimulus, and is not blocked by staurosporine. During the second (mediation) phase, PKC-mediated phosphorylation is mandatory; this phase occurs during the early period of sustained occlusion, and is blocked by staurosporine.

This concept has not, however, been confirmed by all laboratories studying the role of PKC in ischemic preconditioning. For example, Mitchell and colleagues in the rat model (to be discussed in detail later) abolished the protective effect of ischemic preconditioning even when staurosporine was given prior to the preconditioning ischemia and washed out before the sustained ischemic insult [11]. Thus, even if PKC is believed to play a role, there is disagreement regarding the timing of PKC activation. The fact that Yang et al. [53]and Mitchell et al. [11]performed their studies in different species may be an explanation for these discrepancies. On the other hand, it is somewhat hard to believe that such a fundamental protective phenomenon as ischemic preconditioning is occurring via different mechanisms in different species.

In a study performed by Sandhu et al., PKC inhibitors chelerythrine and polymyxin B (introduced just before the 30 min ischemia) only partially blocked the protective effect of ischemic preconditioning induced by one cycle of intermittent ischemia/reperfusion, and did not affect protection induced by three cycles. These results indicate that PKC activation, while contributing to myocardial protection in the one-cycle model, is not a prerequisite in the three-cycles preconditioning model, and implicates the participation of other factors [57]. The one-cycle data presented by Sandhu et al. does not support the previous observation that polymyxin B completely abolishes the protective effect of ischemic preconditioning [8]. The difference in the doses of polymyxin B in studies with partial versus complete loss of protection does not appear to explain the discrepancy, since in the study with partial inhibition the dose of polymyxin B, selected based on its volume of distribution, was reportedly capable of inhibiting more than 90% of PKC present in the heart [57].

Using the in vivo rabbit model, our laboratory tested the PKC hypothesis of ischemic preconditioning by directly measuring the subcellular distribution of PKC in control, preconditioned and sham operated hearts [56]. The preconditioning stimulus consisted of two 5 min episodes of transient regional ischemia each separated by 5 min of reperfusion. Samples were obtained immediately after the preconditioning protocol, or after 10 min into what would be the sustained ischemic insult. PKC was assayed in cytosolic, nuclear and membrane fractions using a radioisotope-based biochemical technique. There was no difference in the subcellular distribution of PKC in hearts sampled immediately after the last episode of preconditioning ischemia/reperfusion when compared with time-matched (nonischemic) controls (Fig. 1A). By 10 min into what would be sustained ischemia, there was a strong trend toward PKC translocation to the nuclear and the membrane fractions in both non-preconditioned controls and preconditioned groups versus nonischenic sham-operated rabbits. Importantly, however, no difference in the subcellular profile of PKC was observed in time-matched groups of preconditioned vs. non-preconditioned hearts. In other words, we failed to demonstrate PKC translocation with 5 min of intermittent preconditioning but, with a more prolonged (i.e. 10 min) sustained ischemic insult PKC does begin to redistribute from the cytosol into the particulate compartment. Our results are in good agreement with the observations of Pongo and colleagues, indicating that in isolated rabbit heart only sustained occlusion of 20 min duration resulted in a significant PKC translocation from the cytosol into the membranes compartment. A preconditioning protocol (i.e. three episodes of ischemia each separated by 5 min of reperfusion) did not induce PKC translocation: rather, an opposite effect (a decrease in its activity) was observed in both the cytosol and the membranes. The authors concluded that, in contrast to the PKC hypothesis, decreased, rather than increased PKC activity might be a factor contributing to the protection rendered by ischemic preconditioning [58]. Lasley et al. came to the same conclusion, when they observed that the PKC inhibitor bisindolylmaleimide significantly reduced infarct size in pentobarbital anesthetized rabbits subjected to 45 min of sustained ischemia [59].

Ping and colleagues employed both a biochemical isoform-nonspecific assay for PKC and an immunoblotting technique. The authors detected ten PKC isoforms in adult rabbit heart. Using the isoform-nonspecific biochemical assay, they found no evidence of PKC translocation following classic in vivo preconditioning. However, when immunoblotting was applied, the authors noticed translocation of PKC and [13]. As discussed previously, this observation is tempered by the question of whether these isoform-selective changes in the subcellular distribution of PKC occurred in myocytes or other cardiac cells. In addition, the authors did not separate the particulate fraction into nuclear vs. membrane components, so the precise site of translocation is not clear.

Since ischemic preconditioning is a receptor-operated phenomenon, involving, in the rabbit model, both adenosine A₁- and -adrenoreceptors, several laboratories attempted to mimic preconditioning by stimulating these receptors. Adenosine and its analogues showed a clear protective effect against sustained ischemic insult, while its antagonists increased the infarct size and/or abolished the protective effect of ischemic preconditioning [4, 60–62]. -Adrenergic agonists (i.e. norepinephrine and phenylephrine) were also capable of inducing protection in the same ischemia model [63–65]. The protective effect of phenylephrine was blocked by both polymyxin B and the adenosine receptor blocker 8-(p-sulfophenyl)theophylline. Based on these observations it was suggested that pharmacological preconditioning is relevant to its classical ischemic version, and PKC activation represents a key event [63, 66]. However, no PKC activity measurements were performed in most of these studies, and this conclusion is solely based on indirect, isoform non-specific pharmacologic studies.

The original ‘PKC hypothesis’ implies that phosphorylation of a target protein following PKC activation is an important step in rendering the heart resistant to sustained ischemic insult. Although PKC substrates are poorly characterized, it is known that these enzymes phosphorylate proteins containing the sequence X-Arg-X-X-Ser/Thr-X-Arg-X [14]. Targets may include myofibrillar proteins, ion channels and MARCKS (myristoylated alanine-rich C kinase substrate) [67]. Given this list of PKC substrates, one may suggest that changes in their structure (i.e. phosphorylation/dephosphorylation) may have significant impact on cardiac cells. In fact, Armstrong et al. demonstrated that the protein phosphatase inhibitor, fostriecin, reduced the rate of development of ischemic contracture and loss of viability in isolated cardiomyocytes during simulated sustained ischemia, thus mimicking the protective effect of ischemic preconditioning [68]. This observation indicates that manipulations downstream of PKC (i.e. modification of protein structure) may play an important role in cardiac protection.

	8 Studies in rats

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
Speechly-Dick and colleagues showed that in the in vivo ischemia model, the PKC inhibitor chelerythrine, administered after a preconditioning stimulus (i.e. prior to sustained ischemia), abolished protection conferred by ischemic preconditioning, and caused an increase in infarct size. In the same study the PKC stimulator 1,2-dioctanoyl-sn-glycerol, also administered prior to sustained ischemia, significantly reduced infarct size. Chelerythrine and the vehicle DMSO had no effect in non-preconditioned rats [10]. Similar results from our laboratory indicated that another PKC inhibitor – calphostin C – also abolished the protective effect of ischemic preconditioning and increased infarct size compared to preconditioned animals. In this study, the PKC inhibitor was administered twice; i.e. before preconditioning ischemia, and 3 min before the onset of sustained coronary occlusion [69]. Thus, regardless whether or not PKC inhibitors were present during the preconditioning stimulus, administration prior to sustained ischemia abolished the protective effect of ischemic preconditioning. These studies may be considered as positive or consistent with the ‘PKC hypothesis’ of preconditioning. However, no direct measurements of PKC activity and/or subcellular distribution were performed. Whether the effects of these particular pharmacological agents are solely attributed to PKC, or are due to other as-yet unknown effects, remains unanswered. For example Li and Kloner reported partial inhibition of preconditioning in the rat model with nonspecific PKC blocker H-7, also known to inhibit PKA and PKG [70, 71].

Confirmatory results have been reported in the isolated rat heart model of regional ischemia: Bugge and Ytrehus found that the PKC antagonists polymyxin B and chelerythrine blocked the protective effect of ischemic preconditioning [72]. The authors also attributed these effects to protein kinase C inhibition, but, unfortunately, no measurements of PKC activity and/or subcellular distribution were performed.

A large body of data, both supporting and contradicting the PKC hypothesis, has been obtained in the isolated rat heart subjected to sustained global ischemia. In this model, functional recovery during reperfusion is typically chosen as the index of damage caused to the heart by sustained ischemia, and to evaluate the protective effect of ischemic preconditioning. For example, Mitchell et al. showed that preconditioning with 2 min of transient ischemia and 10 min of reperfusion significantly increased functional recovery at 40 min after relief of a 20 min period of global ischemia. The PKC inhibitors chelerythrine and staurosporine abolished this protective effect, while the diacylglycerol analogue 1-stearoyl-2-arachidonoyl glycerol mimicked the benefits of ischemic preconditioning. Moreover, immunoblotting for PKC isoforms showed that the two major isoforms in rat heart, PKC and PKC, both translocated during brief episodes of transient ischemia from the cytosol into the membrane and the nuclear compartments [11]. The authors also suggested that PKC translocation upon transient ischemia is labile and can be reversed. This seems to be an attractive addition to the ‘PKC hypothesis’, since constitutive PKC upregulation may cause so-called ‘PKC syndrome’ (i.e. hypertension, atherogenesis, hypercoagulability, insulin resistance, and cancer promotion) [73].

In a study performed by Hu and Nattel two 5 min periods of ischemia followed by 10 min of reperfusion preserved LV developed pressure following 30 min of global ischemia. Pretreatment with PKC inhibitors H-7 and bisindolylmaleimide blocked ischemic preconditioning, while the PKC activator PMA mimicked its protective effect. Based on their results, the authors concluded that PKC stimulation renders the heart resistant to a later sustained ischemic insult [74].

Other results using this model have, however, been inconclusive. Tosaki et al. reported that ischemic preconditioning decreased the incidence of reperfuson arrhythmias, and the PKC inhibitor calphostin C, in doses of 200 and 400 nM, caused a further decrease in their incidence in preconditioned hearts. Thus, in apparent contrast to the ‘PKC hypothesis’ the protective effect of ischemic preconditioning was amplified by the addition of the PKC inhibitor. Interestingly, however, the attenuation of the protective effect of ischemic preconditioning was observed when a very high dose of calphostin C (i.e. 800 nM) was employed. A similar biphasic, dose-dependent pattern was seen with recovery of left ventricular pressure. Although the authors found no protection with preconditioning per se upon recovery of left ventricular function, addition of calphostin C in the concentration of 200–3400 nM augmented, while a concentration of 800 nM inhibited, postischemic functional recovery [75].

In this study, physiologic measurements were accompanied by direct biochemical assessment of PKC activity and its subcellular distribution using the Amersham PKC assay kit. Four cycles of preconditioning ischemia resulted in an increase in PKC activity in both cytosolic and particulate compartments to the same extent. This is a surprising finding, since the original PKC theory predicts only PKC translocation (i.e. redistribution of its activity) rather than a net increase in total amount and/or activity in response to brief ischemia. The authors further reported that all concentrations of calphostin C inhibited PKC in a dose-dependent manner. Thus, in the same preparation, PKC inhibition was shown to have a biphasic effect on arrhythmias and left ventricular function: results obtained with 200 and 400 nM of calphostin C contradicted the PKC theory, while those seen with 800 nM supported the concept [75].

In a study performed by Chen and colleagues, ischemic preconditioning with four cycles of 5 min ischemia/reperfusion significantly improved postischemic functional recovery of the isolated rat heart. Functional recovery was also improved by dioctanoyl glycerol but not by PMA [76]. Thus, as both agents can induce PKC translocation, these results raise doubts as to whether subcellular redistribution of PKC is protective. On the other hand, chelerythrine abolished the protective effect of ischemic preconditioning and blocked functional improvement induced by dioctanoyl glycerol, results that fit the PKC hypothesis. Inconclusive results were also obtained in isolated rat hearts subjected to hypoxia and low-flow ischemia. Brief episodes of hypoxic perfusion (10% of control values) protected isolated rat hearts from sustained no-flow ischemic insult [77]. However, polymyxin B did not alter functional recovery of non-preconditioned hearts subjected to more prolonged low-flow ischemic stimulus [78]. These results indicate that PKC activation and/or translocation (if any) during low-flow ischemia does not underlie the protective effect achieved with brief episodes of hypoxic perfusion, and thus imply that PKC is not the sole mediator of this phenomenon.

While several of the above mentioned studies may be considered as partially ‘positive’ [75, 76, 78], ‘negative’ results have also been obtained in the same isolated rat heart global ischemia model. For example, Galañanes and colleagues, using immunoblotting, were not able to demonstrate translocation of PKC and PKC in the isolated rat heart preconditioned with two brief cycles of ischemia and reperfusion [55]. Recently Moolman and colleagues reported that PKC inhibitors chelerythrine (10 mM) and bisindolylmaleimide (4 mM), given either during the preconditioning protocol or before the onset of sustained ischemia, did not abolish the protective effect of ischemic preconditioning in globally ischemic rat heart. Moreover, the PKC inhibitors were also unable to block the reduction in lactate accumulation caused by ischemic preconditioning [79]. Thus, the conclusion reached by these latter studies is that PKC may not be involved in the mechanism of ischemic preconditioning.

Several receptors have been implicated in the mechanism of ischemic preconditioning in rat model. Mitchell et al. reported that stimulation of -adrenergic receptors with phenylephrine can induce myocardial protection in isolated rat heart, which can be blocked by PKC antagonists (i.e. staurosporine and chelerythrine). They also reported that phenylephrine caused translocation of PKC to the sarcolemma, and suggested that translocation of this PKC isoform is a significant step in preconditioning induced by -adrenoagonists [11]. Moreover, Wilson et al. demonstrated that in rat hearts -adrenergic stimulation in vivo caused significant translocation of another PKC isoform, i.e. PKC (not PKC) [80]. These results indicate that -adrenergic stimulation may trigger translocation of different PKC isoforms in vivo vs. in vitro settings. However, Bugge and Ytrehus did not confirm that -adrenoreceptors are involved in cardiac protection, and reported that blockade of -adrenoreceptors with phenoxybenzamine did not abolish the protective effect of ischemic preconditioning in the isolated rat heart [72]. Recently it has been reported that protection induced by bradykinin and endothelin-1 in the isolated rat heart model was effectively blocked with chelerythrine [81, 82]. Thus, several endogenously produced factors may be involved in heart protection; however, the exact mechanism, and the required combination of these factors remain unclear.

	9 Studies in dogs

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
Studies in dogs have also yielded controversies regarding the involvement of PKC in ischemic preconditioning. In our studies, four 5 min episodes of transient ischemia, each separated by 5 min of reperfusion, significantly limited infarct size caused by a 1 h sustained coronary occlusion. Two PKC blockers, H-7 and polymyxin B, infused before and throughout the preconditioning stimulus and early into sustained ischemia, were unable to abolish the protective effect of ischemic preconditioning. Moreover, we sought to detect PKC translocation during preconditioning by staining tissue samples with a fluorescent probe specific for active PKC, and by radioisotope-based biochemical assay (Amersham PKC Assay Kit). When fluorescence microscopy technique was applied, we observed no change in the intensity or location of fluorescence staining after either one or four 5 min episodes of brief preconditioning ischemia/reperfusion. Similarly, with the radioisotope-based biochemical technique, we were also unable to detect any change in its subcellular distribution in preconditioned vs. control dogs at the end of the preconditioning regimen. When hearts were sampled at 10 min into what would be the sustained occlusion, PKC activity in the particulate fraction was increased in both control and preconditioned groups when compared with dogs that received preconditioning or no intervention alone. Importantly, however, there was no difference in the subcellular distribution of PKC between matched control and preconditioned hearts sampled at either time point. Our positive control (PMA) did, however, yield the expected activation and redistribution of PKC between cytosolic and particulate fraction with both methods. These results fail to support the hypothesis that PKC translocation during brief episodes of preconditioning ischemia is an important factor in the mechanism of ischemic preconditioning in the dog model [49].

In marked contrast to our findings, Kitakaze et al. using the same canine model, reported that polymyxin B, administered only before and during the preconditioning stimulus (not into sustained occlusion), and at a lower dose, completely abolished the protective effect of preconditioning [83]. Contradictory results were also reported regarding PKC activity. In both studies the same assay kit from Amersham was employed. In our experiments we were unable to detect PKC translocation with preconditioning. However, Kitakaze et al. observed significant PKC translocation with preconditioning. This effect was noticed when the assay mixture contained all co-factors; (i.e. lipids and calcium ions) and thus presumably detected all PKC isoforms (conventional, novel and atypical). In contrast, when calcium ions were omitted from the system, the authors were unable to demonstrate PKC translocation. Based on these results, Kitakaze and colleagues concluded, that only conventional Ca-dependent PKC isoforms are translocated with ischemic preconditioning. In the absence of calcium Kitakaze et al. further observed a significant decrease in PKC activity in the cytosolic fraction, thereby indicating that the majority of PKC in this fraction is represented by conventional isoforms. In contrast, in our experiments, under the same conditions we did not observe a decrease in PKC activity in the cytosolic fraction, suggesting that the majority of PKC in the cytosolic fraction is represented by calcium-independent isoforms. When both calcium ions and lipids were omitted from the assay mixture, Kitakaze and colleagues reported no statistically significant changes in PKC signal in the membrane fraction from the ischemic LAD bed, and their calcium- and lipid-independent blank comprised 70 to 85% of what was detected when both co-factors were present in the assay system. In contrast, in our experiments, calcium- and lipid-independent blanks in the particulate fraction from the LAD bed averaged 7 and 9% of the total PKC activity in the control and preconditioned groups respectively.

Our results therefore suggest that the majority of PKC in dog heart is represented by calcium-independent isoforms. This is in agreement with Steinberg et al., who reported that conventional calcium-dependent isoforms are not expressed (or expressed at negligible levels) in adult dog heart: rather, only the novel calcium-independent PKC and atypical PKC were clearly detected [17]. Moreover, we failed to observe PKC translocation during the preconditioning protocol [49]. Data published by Kitakaze and colleagues indicate that the predominant PKC isoforms in canine heart are calcium-dependent, and these isoforms undergo extensive translocation with ischemic preconditioning [83]. At present, there is no explanation for these discrepancies: further investigation using biochemical isoform-specific methods would be required to resolve this issue.

Several conflicting results regarding possible implication of -adrenoreceptors have been reported, with no direct proof of any PKC involvement [84, 85].

	10 Studies in pigs

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
Vogt and colleagues reported that the PKC inhibitors staurosporine and bisindolylmaleimide, administered by intramyocardial infusion, failed to block ischemic preconditioning in the in vivo pig ischemia model, while the adenosine A₁ receptor antagonist cyclopentyltheophylline effectively abolished it. The PKC activator PMA was also able to abolish the protection induced by ischemic preconditioning, while, paradoxically, in non-preconditioned animals, the PKC inhibitor bisindolylmaleimide exerted local protection [86]. This study suggests a key role for adenosine A₁ receptor activation in ischemic preconditioning, and concludes that PKC activation is not involved in its protective effects in the swine model. In contrast to the ‘PKC hypothesis’, these data indicate that PKC inhibition, rather than activation, may protect the heart from a sustained ischemic insult. Similarly, Vahlhaus and colleagues found that, in the pig, staurosporine infused before and during brief preconditioning ischemia did not prevent the protective effect of ischemic preconditioning, again arguing against the involvement of PKC in preconditioning in this model [87]. However, Vahlhaus and colleagues recently provided evidence showing that combined administration of the PKC inhibitor staurosporine and the tyrosine kinase inhibitor genistein abolished the protective effect of ischemic preconditioning in the pig, while either agent alone had no effect on preconditioning-induced cardioprotection [88]. Interpretation of these latter results may be confounded by the fact that genistein, in addition to inhibiting tyrosine kinases, also interferes with the activation of phospholipase C at the G-protein level, and is a competitive antagonist of adenosine receptors, both factors recognized to be important in ‘triggering’ the protection achieved with brief antecedent ischemia [89–91]. Nonetheless, these studies suggest that PKC activation is not a prerequisite for preconditioning in swine, and may implicate the involvement of other protein kinases.

	11 Studies in humans

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
Speechly-Dick and colleagues subjected human right atrial trabeculae to a preconditioning stimulus consisting of 3 min of the simulated ischemia (i.e. hypoxic substrate-free superfusion combined with pacing at 3 Hz) followed by 7 min of reperfusion, and employed recovery of contractile function after 90 min of simulated ischemia as their end point. Contractile recovery was significantly improved with both preconditioning, and exposure of the trabeculae to the PKC activator 1,2-dioctanoyl glycerol, when compared with the non-preconditioned group [92].

The role of PKC in ischemic preconditioning has also been assessed in cultures of pediatric cardiomyocytes obtained during surgery from Tetralogy of Fallot patients. Ischemia was simulated by flushing cultures with 100% nitrogen followed by low-volume anoxia, and injury was assessed by monitoring cellular ability to extrude Trypan blue dye from the intracellular space [93]. A 20 min period of low volume anoxia followed by 20 min of normoxia protected the cardiomyocytes from a later 90 min sustained ischemic insult. Low-volume anoxic preconditioning also was mimicked by brief exposure to phorbol ester (PMA), and was inhibited by the PKC antagonist calphostin C. The authors also performed measurements of PKC activity by in situ phosphorylation of a PKC-specific peptide. PKC-mediated phosphorylation was increased both with preconditioning, and PMA treatment, and was abolished by calphostin C. By employing an isoform-nonspecific fluorescent antibody to PKC, the authors were further able to demonstrate an increase in fluorescence in the cell membranes and in the perinuclear area [94]. As the antibody recognizes sequences in the catalytic site of the PKC molecule, this methods demonstrates that the catalytic PKC structure was indeed present, but does not provide information regarding the integrity of the entire PKC moiety or the activity status of the enzyme. It is also important to note that the preconditioning stimulus and/or adenosine exposure in the above-mentioned study was of 20 min duration. It is well-established that in rat, rabbit and dog, 10 min of ischemia causes PKC translocation [49, 56, 95]. Thus, the changes in PKC activity and/or subcellular distribution seen with 20 min of anoxia may be a secondary phenomenon not directly relevant to preconditioning-induced protection.

Several studies have shown pharmacological preconditioniong of human myocardium. Ikonomides et al. demonstrated that, in pediatric cardiomyocytes, PKC is translocated following 20 min exposure to adenosine, and its activity, determined as the in situ phosphorylation of PKC-specific peptide, is also increased [94]. Cleveland et al. reported that in the isolated human trabeculae the protective effect of ischemic preconditioning can be abolished by -adrenergic blockade and/or PKC inhibition. PKC inhibition with chelerythrine also abolished the protection induced by stimulation of -adrenoreceptors [96]. Since these protocols were performed in in vitro settings and employed non-traditional preconditioning stimuli, their relevance to in vivo ischemic preconditioning is not clear.

	12 Current problems in PKC research

Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 
The protective phenomenon of classical ischemic preconditioning has been unequivocally demonstrated in rats, rabbits, dogs and pigs. In contrast, the role of PKC in this protection is controversial in all of these species. Importantly, however, based on the available information, we can neither unanimously accept nor completely reject the PKC hypothesis. Most of the results in support of the PKC hypothesis were obtained using pharmacological approach; i.e. by administering PKC inhibitors and/or activators. The disadvantages of this strategy were recently summarized in a review published by Brooks and Hearse; almost every inhibitor has ancillary effects and, for some, their specificity and selectivity are questionable [97]. A further limitation of pharmacological studies is that they are isoform-nonspecific. Unfortunately, the same objection is also applicable to all currently available biochemical techniques. With regard to isoform specificity, immunoblotting is the one approach that can be considered as a valuable alternative to pharmacological and biochemical methods. The data provided by immunoblotting is, however, semiquantitative at best, and provides no information regarding the activity of the PKC isoforms.

Recently Gray et al. successfully employed isoform-specific, pharmacological inhibition of PKC. In a cell culture model of hypoxic preconditioning, they found that a specific inhibitor of PKC (i.e. -VI-2 peptide) abolished hypoxic preconditioning and the protection induced by phorbol ester in this particular model [98]. Isoform-specific modifications were also successfully employed in apoptosis-related studies: it was shown that classic and atypical isoforms are anti-apoptotic, while the novel isoforms promote apoptosis [99–102].

The role of PKC in ischemic preconditioning has been evaluated in a diverse array of models, species and protocols. This extreme diversity in experimental design complicates comparisons among studies. For example, data obtained in cultured cardiomyocytes and/or isolated myocardial strips may not be directly relevant to the results obtained in classical ischemic preconditioning protocols, using in vivo models, in which myocardial infarct size is the end point. Also, some of the discrepancies reported in the in vivo rabbit model may be due to different methods of anesthesia (e.g. pentobarbital alone vs. combination of ketamine and xylazine, etc.) [9, 56], and by employing conscious vs anesthetized rabbits [9, 13, 56]. Moreover, global ischemia (usually employed in isolated perfused heart models) appears to differ from regional ischemia in vivo with regard to PKC activation and translocation. This latter point is well-illustrated by data from our laboratory (Fig. 1). In the in vivo rabbit we found no difference in the subcellular distribution of PKC in response to brief preconditioning ischemia (Fig. 1A): at least 10 min of sustained ischemia was required in order to translocate PKC from the cytosolic to the membrane and/or nuclear compartment [56]. However, in globally ischemic rabbit heart the time course of PKC translocation was accelerated, and could be detected at 2 min into sustained ischemia (Fig. 1B) [103]. This striking difference in the time course of PKC translocation may perhaps reflect fundamental pathophysiologic differences between models. For example, with in vivo regional ischemia, the heart is exposed to hemodynamic factors, electrical activity is preserved, and there is still a certain amount of collateral flow, while in the isolated heart subjected to global ischemia, the entire muscle is rendered ischemic, the heart is essentially quiescent, and is not exposed to hemodynamic factors.

As discussed above, we believe there is a need for biochemical, isoform-specific assays. Such assays would allow quantitation of selective PKC isoforms in different subcellular compartments and, if developed, this method, combined with immunoblotting, has the potential to become a valuable tool in PKC studies. We believe it is only through the application of isoform- and cell-specific assays that the role of PKC in ischemic preconditioning can be conclusively resolved.

Time for primary review 22 days

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Top Abstract 1 Introduction 2 The function of... 3 Cardiac PKC isoforms... 4 The concept of... 5 Measurement of PKC... 6 PKC and ischemic... 7 Studies in rabbits 8 Studies in rats 9 Studies in dogs 10 Studies in pigs 11 Studies in humans 12 Current problems in... References

 

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