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(Circulation Research. 2000;86:926.)
© 2000 American Heart Association, Inc.

MiniReviews

Ischemic Preconditioning in Isolated Cells

Michael S. Marber

From the Department of Cardiology, St Thomas’ Hospital, King’s College London, London, England.

Correspondence to Dr Michael S. Marber, Department of Cardiology, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH, UK. E-mail mike.marber{at}kcl.ac.uk

Key Words: ischemic preconditioning • signaling • cytoprotection • isolated cardiomyocytes

	Introduction

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Ischemic preconditioning describes the increased resistance to myocardial infarction that follows short sublethal periods of ischemia. After the ischemia that triggers preconditioning, there are 2 phases of protection: an early (short-lived) phase termed early or classic preconditioning¹ and a late (more prolonged) phase termed late preconditioning or the second window of protection.² The purpose of the present minireview is to highlight the contributions being made to our current understanding of early preconditioning by models based on isolated cardiac cells.

Within the preconditioning literature are disparate findings usually explained by variations in species, maturity, preconditioning trigger, anesthetic, and/or choice of end point. This lack of generality is a particular problem with cell-based models. Variability in the circumstances of the trigger, simulation of ischemia, in vitro maintenance conditions, cell type, and species of origin result in innumerable combinations and permutations (see online data supplement at http://www.circresaha.org), making findings difficult enough to compare between cell models let alone between these models and preconditioning in the whole heart. Given these drawbacks, why are an increasing number of preconditioning investigators adopting a cell-based approach?

	Relative Merits of Cell-Based Models of Preconditioning

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Little controversy surrounds the surface receptors able to trigger preconditioning. In the whole heart, their successful pharmacological manipulation can be verified by alterations in vascular resistance, rate and strength of contraction, and atrioventricular conduction. However, attention has now shifted to intracellular signaling pathways, and the specificity of pharmacological agents is diminished, their effects on physiology are less certain, and their costs are greatly increased. The cell-based models overcome these disadvantages through a small volume of distribution, through an ability to manipulate signaling proteins by the introduction of cDNAs, antisense RNA, recombinant protein, and interfering peptides, and through the interrogation of altered signaling cascades and their consequences within a homogeneous cell type. However, these advantages are at the expense of a cell phenotype that differs from the intact heart and cannot be subjected to true ischemia/reperfusion. Thus, mechanisms may not reflect those in vivo. The advantages and disadvantages are listed in an online data supplement (see http://www.circresaha.org). The remainder of the present review focuses on the different cell-based models and their contributions to the preconditioning field.

	Cell-Based Models of Preconditioning Using Immature Cardiomyocytes

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Mature (adult) cardiac myocytes die or dedifferentiate in long-term culture and are resistant to classic transfection. Thus, preconditioning models based on immature or dedifferentiated cardiocytes were the first described^{3 4} and are still favored by many investigators (see Table 1). The cells used fall into 3 types: (1) embryonic, usually chick myocytes; (2) neonatal, usually rat myocytes; and (3) adult cells, usually human, that have been "dedifferentiated," resulting in lost rod-shaped morphology and recrudescence of mitosis.

View this table: [in this window] [in a new window]   Table 1. Models of Preconditioning in Immature Cardiocytes

The popularity of immature cardiocytes is based on their familiarity as a paradigm of hypertrophy and the similarities that exist between the signaling processes of hypertrophy and preconditioning. However, documented differences between mature and immature myocytes require proposed cell-based models to faithfully recapitulate key features of ischemic preconditioning. These features include initiation by simulated ischemia, the temporal relationship between initiating and lethal simulated ischemia, the involvement of ligands to G-protein–coupled receptors, and protein kinase C (PKC) dependence. Despite wide variations in species of origin and experimental detail, models based on immature cells fulfil these criteria. In common with ischemic preconditioning in the whole heart are temporal associations between sublethal and lethal ischemia,^{5 6} ligands able to trigger protection,⁷ PKC inhibitors able to block protection,⁸ and end points such as intracellular protein release and trypan blue uptake,³ which are more indicative of cell death by necrosis than apoptosis.

Triggers for Preconditioning
Immature cardiocytes provide confirmatory evidence that early^{3 8 9 10} and late⁹ preconditioning exists in human cardiomyocytes but not in human endothelial cells.¹⁰ Furthermore, there is sufficient adenosine release to confer protection to "naïve" cells, an effect mimicked by a nonselective adenosine agonist and blocked by a nonselective adenosine antagonist or PKC inhibitor.⁸ This pattern of adenosine-triggered hypoxic preconditioning is identical to that seen in cultured chick cardiomyocytes.¹¹ However, this model has the advantage of permissive transfection with efficiencies of 40% with calcium phosphate.¹² Indirect evidence suggests that protection triggered by hypoxic preconditioning is A₁^{11 13} and A3^{13 14} adenosine receptor dependent. In contrast, an A2a-selective agonist during brief hypoxia aggravates injury, whereas an antagonist is protective on its own and augments the protection seen with the nonselective adenosine agonist R-phenylisopropyladenosine,¹⁴ suggesting that preconditioning could be even more protective with concomitant blockade of the adenosine A_2a receptor. This observation is further reinforced by transient transfection of the cDNAs of the human A₁ and A₃ receptors. Monolayers expressing these receptors are more resistant to lethal hypoxia but more sensitive to the protective effects of sublethal/preconditioning hypoxia.¹² This suggests that adenosine receptor occupancy is protective during both lethal and sublethal hypoxia, reflecting findings in the intact heart as well as findings for other G-protein–coupled receptor agonists in isolated immature cardiocytes.¹⁵ Another interesting aspect of these studies is that atrial myocytes, deficient in endogenous A₃ receptors, can be rendered A₃ responsive and resistant to simulated ischemia by forced expression of the human A₃ receptor.¹³ Moreover, protection initiated by the A₃ receptor seems longer lasting than that initiated by the A₁ receptor,¹³ and this may in part be explained by differential coupling of A₁ to phospholipase C and A₃ to phospholipase D.¹⁶

Evidence in these models favors endogenous adenosine as the initiator of preconditioning, but protection can also be triggered "directly" by morphine through opioid receptors.⁷ Under this circumstance, protection is prevented by ATP-sensitive K⁺ (K_ATP) channel blockade before, but not necessarily during, lethal hypoxia.⁷ Although it is apparently controversial, there is similar evidence in the intact heart¹⁷ and emerging evidence in isolated adult cardiocytes^{18 19 20} indicating that the mitochondrial K_ATP channel may not be the end effector of protection. This would be in keeping with observations in embryonic myocytes, in which the trigger for preconditioning during sublethal hypoxia involves the mitochondrial export of superoxide generated at cytochrome b-c₁ of complex III of the electron transport chain.²¹ Acetylcholine-triggered preconditioning in this model also requires mitochondria-derived superoxide, and this is also dependent on the opening of mitochondrial K_ATP channels.^{22 23} The hypothesis that the opening of mitochondrial K_ATP channels causes partial collapse of the mitochondrial potential and therefore "functional" uncoupling of electron transport with increased superoxide generation is attractive, potentially unifying and merits further attention.

Signaling Pathways Leading to Protection
The experiments above confirm and extend the knowledge gained in the intact heart of the ligands that can lead to preconditioning. However, the pathways that lie distal to adenosine (or similar) receptors are more controversial. Because hypoxic preconditioning in immature cardiocyte-based models of preconditioning, in common with preconditioning in the intact heart, is blocked by pharmacological inhibition of PKC,^{4 8 24 25} these models have been used to further explore the importance of individual PKC isotypes.

PKCs constitute a catalytic subunit linked by a flexible hinge region to a regulatory domain containing an amino acid sequence nearly identical to that used to recognize substrate. In the model proposed for PKC regulation, this pseudosubstrate site allosterically prevents the binding, and therefore phosphorylation, of target proteins.²⁶ On activation, a conformational change is envisaged that opens up the hinge region and dissociates the pseudosubstrate domain, freeing the substrate binding site and also exposing residues that bind to specific receptors for activated C kinases (RACKs).²⁷ The RACKs, in turn, are thought to traffic activated PKC isotypes to their correct subcellular location (Figure). Recent evidence is also emerging that other events may modulate PKC function through key phosphorylation events within an activation loop,²⁸ which may allow activation in the absence of translocation.

View larger version (45K): [in this window] [in a new window]   Figure 1. The putative signaling pathways involved in ischemic preconditioning of isolated cells are as follows: (1) A heptahelical transmembrane receptor coupled through its cytoplasmic tail to a heterotrimeric G protein is depicted.54 In cell-based models, protection can be initiated through a number of these receptors3 8 11 20 23 35 36 37 41 42 but can also be blocked if the receptor couples to a G protein with an s subunit14 (see text for details). (2) On activation of the receptor, dissociation of the G protein allows the subunit to activate PKC through a classical phospholipase activation pathway54 and also perhaps through phospholipase-independent pathways via the ß subunits. (3) The increase in diacyl glycerol and perhaps calcium activates the PKC protein by binding to the regulatory domains R1 and R2. Activation involves a conformational change and translocation through a number of PKC binding proteins (see text). (4) The conformational change also results in the pseudosubstrate site at the N-terminus dissociating from the substrate-binding site within the catalytic region (C). Once the pseudosubstrate site has dissociated, the catalytic region is able to bind substrate. Manipulation of PKC binding proteins and the pseudosubstrate site has been used to investigate the isotype dependence of preconditioning (see text for details).24 25 29 38 (5) PKC activation favors opening of the sarcolemmal KATP channel (see online data supplement at http://www. circresaha.org). (6) The manipulations of PKC that open the sarcolemmal channel also seem to favor the opening of a putative mitochondrial KATP channel (see online data supplement at http://www.circresaha.org). Opening the mitochondrial KATP channel results in the formation of superoxide anions.23 Although not directly tested in cell models of preconditioning, the superoxide anions may activate PKC,55 resulting in a positive-feedback loop. Such a loop would help reconcile some of the apparent paradoxes, suggesting that KATP is an initiator rather than a final effector of protection (see online data supplement at http://www.circresaha.org). (7) The activation of PKC has been shown to cause the activation of the p42/44-MAPK pathway, protecting isolated cardiomyocytes from simulated ischemia.44 Similarly, during simulated ischemia, p38-MAPK is activated31 and injures isolated cardiomyocytes.31 33 Preliminary data suggest that PKC and preconditioning inhibit p38 activation.32

In a model of hypoxic preconditioning of neonatal rat cardiomyocytes, PKC and PKC translocate in response to preconditioning, and translocation is associated with protection from subsequent more prolonged hypoxia.^{24 25} Johnson et al²⁹ have previously demonstrated that a peptide corresponding to residues 14 to 21 (V1-V2) of PKC is capable of inhibiting the translocation of PKC. It is thought that a RACK-binding domain exists in the V1-V2 region so that the peptide saturates the appropriate RACK, preventing the translocation of PKC and protection.²⁴

We have adopted a complementary approach by expressing mutant PKC isotypes rendered constitutively active by deletions within the pseudosubstrate domain,²⁵ which prevent the autoinhibition seen in the wild-type molecule (see above). We have shown that active PKC consistently reduces hypoxic injury, an effect not seen with the expression of wild-type PKC.²⁵ These experiments demonstrate that active PKC is able to trigger protection but do not indicate that PKC is the endogenous isotype responsible for protection. The rat neonatal cardiocyte model has also been used to investigate the more distal mitogen-activated protein kinase (MAPK) pathways involved in protection. These experiments are controversial and heavily reliant on the nonspecific p38-MAPK inhibitor SB203580.³⁰ Data from Mackay and Mochly-Rosen³¹ and preliminary data from our group³² demonstrate that p38-MAPK undergoes a period of prolonged activation during lethal hypoxia and that if this kinase is inhibited by SB203580, then injury is reduced. Moreover, the p38-MAPK isotype preferentially activated by simulated ischemia is p38,³² reinforcing the known role of this isotype in mediating cell death within this model.³³

	Cell-Based Models of Preconditioning Using Mature Cardiomyocytes

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Isolated adult cardiomyocytes have been used for almost 2 decades to study cell injury³⁴ despite a rate of attrition as great as 30% in the first 24 hours after isolation.³⁵ Therefore, models largely rely on suspensions of freshly isolated cells. The first, and most widely adopted, model is that described by Armstrong et al³⁶ of lethal simulated ischemia achieved by overlaying rabbit adult cardiomyocytes compacted into a cell pellet with mineral oil (see Table 2). Preconditioning initiated by either a short period of cell pelleting or by suspension in glucose-free buffer shares many features with preconditioning in the intact heart, including the adenosine,^{36 37} PKC,^{36 38} and K_ATP^{20 35 39} dependence of protection.

View this table: [in this window] [in a new window]   Table 2. Models of Preconditioning in Mature Cardiocytes

Triggers for Preconditioning
Preconditioning is initiated in the intact rabbit heart by A₁-selective agonists.⁴⁰ However, in isolated rabbit cardiomyocytes, the relationship is more complex because an A₁/A₂-selective agonist does not substitute for 15 minutes of glucose-free preconditioning, but a nonselective adenosine receptor antagonist during the glucose-free period prevents preconditioning.³⁶ This apparent paradox was resolved in a subsequent study in which 5 minutes of glucose-free incubation, with or without pyruvate, initiated preconditioning, which could be blocked by an A₃- but not A₁-specific antagonist.³⁷ Exclusive initiation through the A₃ receptor was confirmed by triggering preconditioning with a mixed A₁ and A₃ agonist alone or together with an A₁ but not an A₃ antagonist.³⁷ The finding of A₃-initiated protection is consistent with immature cardiocytes and isolated adult porcine cardiocytes.^{12 14 41} Protection can also be initiated through opioid receptors in the rabbit cardiomyocyte pelleting model,⁴² metabolic inhibition in adult rat cardiocytes,³⁵ and chloride channels during cell swelling–induced preconditioning in adult rabbit cardiocytes.⁴³

Signaling Pathways Leading to Protection
The PKC isotype dependence and MAPK pathways leading to preconditioning have also been examined in adult cardiocytes. Ping et al⁴⁴ confirmed observations made in vivo by transfecting adult rabbit cardiocytes in culture with adenoviral vectors encoding PKC. In this extensive study, LDH release during and after 6 hours of simulated ischemia was diminished in cells overexpressing wild-type PKC. This virus also increased p44-MAPK and, to a lesser extent, p42-MAPK activity, an effect that (together with increased resistance to ischemia) was abolished by an upstream MAPK inhibitor or by cotransfecting dominant-negative PKC,⁴⁴ suggesting that PKC mediates protection through enhancing the activation of p42/44-MAPK.⁴⁴ This PKC-isotype dependence is in broad agreement with findings in pelleted adult rabbit cardiomyocytes, in which preconditioning is abolished by the PKC V1-V2.³⁸ In this model, in common with immature cardiocytes, there is a period of early and prolonged p38-MAPK activation for 60 minutes.⁴⁵ In cell pellets that have been preconditioned, this activation is significantly enhanced at the 30-minute, but nonsignificantly diminished at the 60-minute, time point.⁴⁵ The nonspecific p38-MAPK inhibitor SB203580 reduced p38-MAPK phosphorylation and also sensitized cells to simulated ischemia, suggesting that p38-MAPK activation is protective, but this did not correlate with the phosphorylation/translocation of the downstream substrate hsp27.⁴⁵ Furthermore, it is not clear which p38 isotype contributed to the enhanced activity at 30 minutes. Therefore, it is possible that these results⁴⁵ may still be consistent with the proposed detrimental effect of p38 activation in neonatal cardiocytes.^{31 32 33}

Experimental evidence in mature cardiomyocytes underpins the existence of a distal and separate mitochondrial K_ATP channel. This evidence was the focus of a recent minireview⁴⁶ and is further discussed in an online data supplement (see http://www.circresaha.org).

	Cell-Based Models of Preconditioning in Noncardiomyocytes

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Compared with cardiomyocytes, noncardiac cells are usually more resilient, available, and transfection permissive. In addition, mitosis and transfection with selectable markers allow the formation of stable cell lines. However, their drawback is inconsistent preconditioning. For example, with preconditioning by glucose withdrawal against cell pelleting, adult rabbit cardiomyocytes and differentiated C₂C₁₂ (mouse skeletal muscle–derived) cells precondition, but HEK293 (human embryonic kidney–derived) cells, HIT-TIG (hamster pancreatic islet–derived) cells, and undifferentiated C₂C₁₂ cells do not.⁴⁷ Similarly, although the supernatant from endothelial cells exposed to brief hypoxia is able to precondition ventricular myocytes,^{10 41} the endothelial cells themselves are not protected¹⁰ but have attenuated posthypoxic intercellular adhesion molecule-1 induction.⁴⁸ Despite an inability to initiate preconditioning with simulated ischemia in undifferentiated cell lines, these cells still seem to be protected by the introduction of components of the preconditioning pathway.^{49 50} Therefore, undifferentiated cell lines may still be of use in the molecular dissection of pathways leading to protection.

	Future Directions and Summary

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To an outside observer, it must seem that the preconditioning field has stagnated in K_ATP channels and kinase pathways. The weight of evidence is overwhelming that both contribute to protection, but what is lacking is the detail. The manipulations required to determine this detail are most easily achieved in isolated cells. The work summarized in the present review has begun to examine this detail and to link sarcolemmal receptors with PKC, K_ATP channels, p42/44-MAPK, and p38-MAPK (see Figure). What is now required is further investment in models and tools to complete the task. In particular, a combination of techniques is required to confirm that the mechanistic insights derived from isolated cells reflect the mechanisms in vivo.⁵¹

The concern that the mechanisms of preconditioning may vary with the detail of the model has made many wary of extrapolating findings from cell-based models. However, within the present review, there is a high degree of concordance between the mechanisms underlying preconditioning in different cellular models and those found in the intact animal heart. Ultimately, irrespective of the model, insights into how to bottle preconditioning will need to be tested in the only circumstance that counts, true myocardial ischemia in humans.

This MiniReview is part of a thematic series on Preconditioning, which includes the following articles:

Ischemic Preconditioning in Isolated Cells

Second Window of Preconditioning Clinical Role for the Preconditioning Phenomenon: An Appeal for a Reasoned Approach Myocardial K_ATP Channels in Preconditioning

Roberto Bolli, Editor

Received January 18, 2000; accepted March 29, 2000.

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