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Cardiovascular Research 2006 70(2):274-285; doi:10.1016/j.cardiores.2006.02.011
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Copyright © 2006, European Society of Cardiology

The end-effectors of preconditioning protection against myocardial cell death secondary to ischemia–reperfusion

David Garcia-Dorado*, Antonio Rodriguez-Sinovas, Marisol Ruiz-Meana, Javier Inserte, Luis Agulló and Alberto Cabestrero

Servicio de Cardiologia, Hospital Universitari Vall d–Hebron, Passeig Vall d–Hebron, 119-129, 08035 Barcelona, Spain

* Corresponding author. Tel.: +34 93 4894038; fax:+ 34 93 4894032. Email address: dgdorado{at}vhebron.net

Received 16 December 2005; revised 5 February 2006; accepted 9 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. The effectors of...
 3. The effectors of...
 4. Conclusion
 References
 
Our understanding of the end-effectors involved in preconditioning protection is still very limited. This is partially due to an incomplete knowledge of the mechanisms responsible for acute sarcolemmal rupture and cell death during the first minutes of reperfusion, including the relative roles of hypercontracture-mediated sarcolemmal rupture and mitochondrial permeability transition pore (MPTP) opening-mediated cell death. In the present article, the role of proposed end-effectors of preconditioning protection, defined as molecules directly involved in cell death that are modified by ischemic preconditioning (IP), is examined. IP attenuates hypercontracture-mediated cell death, probably through several mechanisms, including attenuated calpain activation during reperfusion leading to preserved cytoskeletal integrity and accelerated recovery of Na+/K+-ATPase function, but probably also protein kinase G (PKG)-mediated improved calcium handling. The potential role of gap junctions in preconditioning protection is controversial, but the recently discovered mitochondrial localisation of connexin43 seems to play an important role in protection that has not yet been completely defined. Several recent studies suggest that IP can reduce MPTP opening during reperfusion and limit infarct size through this mechanism, although the contribution of this widely accepted mechanism to the infarct size reduction induced by IP in the intact heart needs to be established.

KEYWORDS Myocardial infarction; Hypercontracture; Calpain; Connexin43; cGMP; PKG; Mitochondrial permeability transition; GSK3beta


   1. Introduction
Top
 Abstract
 1. Introduction
2. The effectors of...
 3. The effectors of...
 4. Conclusion
 References
 
Since the protection afforded by brief episodes of ischemia against cell death induced by subsequent prolonged ischemia and reperfusion was originally described in 1986 [1], an enormous amount of information has been generated on the molecular mechanisms involved in the genesis of this phenomenon known as ischemic preconditioning (IP). A large number of stimuli able to trigger a state of increased resistance to ischemic–reperfusion have been identified, and the multiple and interrelated signal transduction pathways downstream of preconditioning triggers have been elucidated to a large extent. However, the end-effectors of preconditioning protection, the last links of the long chain of events that starts with triggers and ends with protection, remain largely unknown.

End-effectors of preconditioning can be defined as molecules that are modified by preconditioning and have a definite and direct role in cell death. The identification of the end-effectors of preconditioning protection has important practical implications because these effectors have a great potential as pharmacological targets. Acting directly on end-effectors holds much more therapeutic promise than trying to induce protection with preconditioning triggers, because triggers, by definition, should be applied before ischemia, which is normally not possible. It can be argued that different pharmacological treatments and postconditioning have been shown able to limit lethal reperfusion injury when applied at the time of reperfusion. However, most of the drugs presently available for this purpose (for example, inhibitors of sarcolemmal Na+/Ca2+ exchange) cannot be used in patients, and although postconditioning has been shown to be feasible as well as probably safe and effective in patients with acute myocardial infarction receiving primary percutaneous coronary angioplasty, it cannot be applied to patients not receiving percutaneous mechanical revascularisation.

In this article, we will briefly analyse the mechanisms of cardiomyocyte death during ischemia–reperfusion and then review the available information on how preconditioning may modify them.


   2. The effectors of cell death during myocardial ischemia–reperfusion
Top
 Abstract
 1. Introduction
 2. The effectors of...
3. The effectors of...
 4. Conclusion
 References
 
In the context of ischemia–reperfusion, cell death can occur in different ways. Some of these ways have in common the rupture of plasma membrane, an undisputed feature of necrotic cell death. Cells can also experience apoptosis, an active, relatively long process without a clearly identifiable point of no return. It is now widely accepted that necrosis and apoptosis are the ends of a continuous spectrum, that the cascades of events leading to necrosis and apoptosis may share multiple elements, that cells can switch between them, and that the final form of cell death depends largely on the conditions [2]. It is also generally accepted that, under most circumstances, cell death secondary to ischemia–reperfusion involves sarcolemmal rupture and occurs during the first minutes after restoration of blood flow and is not followed, in the absence of persistent ischemia, by significant additional cell death during the following hours [3].

2.1 Hypercontracture-mediated sarcolemmal rupture
There is very solid evidence [4] that excessive contractile activation (hypercontracture) plays a very important role in cell death: 1) the occurrence of hypercontracture during the first minutes of reperfusion has been documented in vitro and in vivo (by microcrystal analysis of myocardial segment length); 2) histological analysis demonstrates that reperfused infarcts consist almost exclusively of contraction band necrosis; 3) contraction band necrosis is established during the first minutes of reperfusion; 4) there is a narrow correlation between the magnitude of hypercontracture, of enzyme release, and of contraction band necrosis; 5) there is a close correlation between the time course of hypercontracture and that of lactate dehydrogenase (LDH) release; 6) brief, transient inhibition of contractility during reperfusion results in inhibition of enzyme release, and restoration of contractile activity is associated with LDH release occurring in vivo; 7) prolonged inhibition of contractility until myocytes recover Ca2+ control results in very pronounced infarct limitation. The observation that contractile inhibition prevents sarcolemmal rupture has been reproduced by different laboratories in a variety of models [5–9].

Ca2+ overload and altered Ca2+ handling, with oscillatory cytosolic Ca2+ concentration, play a prominent role in hypercontracture. Ca2+ overload is the consequence of Ca2+ influx through reverse operation of the sarcolemmal Na+/Ca2+ exchanger (NCX) during preceding ischemia and additional Ca2+ through the same mechanisms during the first minutes of reperfusion [10,11]. The reverse operation of the sarcolemmal NCX is the consequence of increased cytosolic Na+ concentration and lasts until restoration of Na+ pump activity normalises it [3,4]. Cytosolic Ca2+ oscillations are caused by the back and forth movement of Ca2+ between cytosol and sarcoplasmic reticulum (SR), and their inhibition with blockers of SR Ca2+ uptake or release prevents hypercontracture. It has been recently described that, under some circumstances, the extreme cell shortening observed in hypercontracture may be the consequence of sustained rigor-bond associated cell shortening rather than being caused by ATP and Ca2+-dependent contractile cycling [12]. Although the relevance of this phenomenon during in vivo myocardial reperfusion has not yet been established, it may be important as it provides a potential connection between the hypercontracture paradigm of reperfusion cell death and the mitochondrial permeability transition theory (see below).

Sarcolemmal rupture is importantly favoured by cytoskeletal fragility, in particular by calpain-dependent hydrolysis of {alpha}-fodrin [13], a filamentous protein that forms a mesh giving mechanical strength to the sarcolemma. Ca2+-dependent activation of calpain occurring at the time of reperfusion not only causes sarcolemmal fragility, but importantly contributes to the dysfunction of the sarcolemmal Na+ pump by degrading ankyrin, the protein that attaches it to the fodrin cytoskeleton, as well as fodrin itself. Delocalisation and dysfunction of the sarcolemmal Na+ pump impairs the normalisation of cytosolic Na+ concentration and closes a vicious circle "increased Na+, increased Ca2+, increased calpain activation, more Na+pump dysfunction, aggravated Na+ overload", etc. [8]. Sarcolemmal rupture causes massive Na+ influx that can propagate to adjacent cardiomyocytes through gap junctions [14].

2.2 Mitochondrial permeability transition pore (MPTP)
Since its original description, MPTP opening has been recognised as an important mechanism of cell death [2]. The available information on the molecular identity, regulation, and conductance of this megachannel and its involvement in different types of cell death has been reviewed in detail elsewhere [2] and will be only very briefly mentioned here. We will discuss in some detail, however, the potential relation between MPTP and the mechanisms of cell death described above.

It is clear that sustained MPTP opening resulting in complete mitochondrial depolarisation, massive mitochondrial swelling, and rupture of external mitochondrial membranes is incompatible with cell life, and that during reperfusion the conditions are present to induce sustained MPTP opening, including Ca2+ overload, oxidative stress, low ATP concentration, and pH normalisation [2]. Induction of MPTP opening kills isolated cells [2,15]. Although MPTP opening in intact cells and tissues is difficult to monitor, analysis of NAD+ obtained in the effluent of reperfused rat hearts indicates that MPTP opening occurs in fact during reperfusion [16]. Evidence of MPTP opening during in vivo reperfusion, and not during preceding ischemia, has been reported in hearts reperfused after 30 min of ischemia by using an ingenious method involving loading cells with radioactive 2-deoxyglucose (DOG) [17,18]. Once phosphorylated in the cytosolic compartment, DOG should not be able to enter the mitochondrial matrix except through open MPTP. With this technique, it has been possible to show that MPTP opening during reperfusion follows a time course parallel to that of intracellular pH normalisation, a finding fully consistent with the powerful inhibitory effect of acidosis on MPTP. Moreover, transgenic mice lacking cyclophylin D, one of the elements necessary for the opening of the multiprotein megachannel, show markedly reduced necrosis when exposed to ischemia–reperfusion [19].

2.3 Hypercontracture and/or MPTP?
An unsolved, yet important, question is the relationship between hypercontracture and MPTP opening as causes of cell death; that is, the elucidation of whether they represent independent death pathways, or which of these events comes first. MPTP opening could lead to rapid sarcolemmal rupture [20], massive Ca2+ overload, and hypercontracture. On the other hand, hypercontracture-mediated sarcolemmal rupture could expose mitochondria to extracellular Ca2+ concentrations and cause MPTP opening. It could also be that MPTP and hypercontracture are alternative mechanisms of cell death whose relative importance depends on the circumstances (Fig. 1).


Figure 1
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  		Fig. 1 Potential relationships between hypercontracture and mitochondrial permeability transition pore (MPTP) as causes of sarcolemmal rupture and necrotic cell death during early myocardial reperfusion. Hypercontracture can cause MPTP opening (gray thick arrows) and vice versa (black thick arrows). ROS: radical oxygen species.

 
On one hand, hypercontracture resulting from excessive Ca2+-dependent contractile activation requires energy, and isolated cardiomyocytes hypercontracted after prolonged and severe energy depletion are metabolically competent as far as they retain membrane integrity [9,21,22]. This indicates that hypercontracture may kill reperfused myocytes before MPTP opening can occur.

However, a second type of extreme cell shortening has been described during reperfusion that can take place in the presence of critically low ATP concentrations. This type of contracture is due to severe rigor-type shortening occurring at the time of reperfusion in cells already shortened by rigor contracture during prior ischemia [12]. This rigor-type, reperfusion-induced contracture ("hyper-rigor") is supposed to be the consequence of impaired recovery of mitochondrial ATP synthesis resulting in slow ATP recovery and prolonged permanence of the increasing ATP concentration within the critical low concentration window at which contracture occurs. In contrast to classical Ca2+-dependent hypercontracture, rigor-type, reperfusion-induced hypercontracture could occur after substantial MPTP opening has taken place, or as a consequence of it.

Nevertheless, available information indicates an important role for energy-dependent hypercontracture even after prolonged periods of ischemia. Although the metabolic competence of the intact myocardium at the time of reperfusion-induced hypercontracture is difficult to determine in vivo, because in intact myocardium hypercontracture normally causes sarcolemmal rupture [3,10], valuable information can be obtained by adding the contraction blocker 2,3-butanedionemonoxime (BDM) to the perfusion media during the first minutes of reflow. This prevents hypercontracture and cell death (LDH release) for the time the drug is present (or definitively, if this period is prolonged enough [8]). Nuclear magnetic resonance (NMR) spectroscopy demonstrates a rapid and virtually full recovery of phosphocreatine (PCr), indicative of metabolic competence, in hearts reperfused in the presence of BDM after 60 min of ischemia, a duration of ischemia that results in massive reperfusion-induced hypercontracture and LDH release in the absence of the drug (unpublished observations). This suggests that in intact rat myocardium reperfused after 60 min of ischemia, hypercontracture is the primary event leading to sarcolemmal rupture.

On the other hand, the hypothesis that hypercontracture is the prime cause of cell death is in apparent contrast to experiments showing protective effects of MPTP inhibitors on infarct size and, more importantly, the protective effect of cyclophylin D gene ablation on post-reperfusion necrosis. Unfortunately, there is a lack of powerful and specific pharmacological inhibitors of MPTP that can be used in intact hearts or in vivo [2,23]. Cyclosporine A (CsA) and sanglypherin are probably the most specific MPTP inhibitors available [2], but their specificity is still suboptimal. In fact, the protective effects of CsA against myocardial infarction were initially ascribed to its powerful inhibitory effect on calcineurin [24], a Ca2+-calmodulin-dependent phosphatase that has effects on SR calcium handling that are not completely understood [25]. In addition, the protective effects of these compounds against MPTP opening in isolated mitochondria and against cell death occurring during in vivo myocardial reperfusion seem to be variable and dependent on the experimental conditions [2].

The most conclusive proof of the involvement of MPTP in reperfusion-induced necrosis comes from studies in mice lacking cyclophilin D [19,26]. Consistent with previous studies assigning an important regulatory role to cyclophylin D on MPTP, heart or liver mitochondria isolated from cyclophylin D– / – animals show a dramatically increased resistance to MPTP opening in response to high Ca2+ concentrations or other stimuli. Two studies have documented a reduction in infarct size after transient (30 min) regional ischemia in brain [26] and heart [19] in these animals. This latter represents the only study in myocardial tissue (able to develop hypercontracture) in which a genetic intervention aimed to prevent MPTP opening reduces reperfusion necrosis. Although several possibilities exist (for example, activation of different proteases), the exact connection between MPTP and sarcolemmal rupture has not been established.

Clearly, additional studies will be necessary to determine the respective contributions of hypercontracture-mediated sarcolemmal rupture and MPTP opening in cell death occurring during reperfusion. A plausible scenario is that both are potential effectors of cell death that take on either the role of a real executioner or an epiphenomenon, depending on the circumstances (Fig. 1).


   3. The effectors of preconditioning protection
Top
 Abstract
 1. Introduction
 2. The effectors of...
 3. The effectors of...
4. Conclusion
 References
 
We believe that using a strict definition of preconditioning end-effector, as described in Table 1, would be of great help in the discussion of the merits of the potential end-effectors thus far proposed. Following these criteria, the present review is focused on molecules directly involved in hypercontracture-mediated sarcolemmal rupture or in MPTP opening that have been described to be modified by IP (Fig. 2). We will limit the discussion to classical, acute IP.


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  		Table 1 Criteria qualifying a molecule as an end-effector of preconditioning protection

 

Figure 2
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  		Fig. 2 Diagram of the mechanisms of cardiomyocyte cell death during reperfusion showing proposed end-effectors of preconditioning (IP) protection; in gray, end-effectors for which available evidence is weaker.

 
3.1 Attenuated Ca2+-dependent hypercontracture
Although it has been known for a long time that IP exerts its protection mainly at the time of reperfusion [27], and although it has been hypothesised that this effect could be mediated by a reduction of hypercontracture-mediated sarcolemmal rupture [28], the evidence obtained of improved Ca2+ handling or protein desensitisation is scant thus far and mainly related to cGMP-dependent protein kinase (PKG) activation.

Recent studies suggest that increasing cGMP may trigger preconditioning protection [29] and that PKG may be important for transmission of preconditioning signals between cytosol and mitochondria [30], but the role of the cGMP/PKG pathway as an effector of classical, acute preconditioning has not been investigated in depth. The cGMP/PKG pathway is severely depressed in cardiomyocytes and endothelial cells reperfused after prolonged ischemia [30–33], and preserving this pathway is protective against reperfusion-induced cell death [34–36]. It has also been shown that at the time of reperfusion, cGMP/PKG is preserved in preconditioned myocardium. IP attenuates the reduction in NO availability during subsequent index ischemia in isolated rat hearts [37], and the marked reduction in the myocardial synthesis of cGMP can be partially reversed by preconditioning [38].

cGMP may protect cardiomyocytes against hypercontracture and sarcolemmal rupture by multiple mechanisms. A prominent one is interference with SR-driven oscillations in the cytosolic Ca2+ concentration. It has been shown that sodium nitroprusside, natriuretic peptides, and cGMP analogues [39,40] induce phosphorylation of phospholamban and thus enhance Ca2+ uptake by the SR. A recent study has solidly demonstrated that stimulation of membrane-bound guanylyl cyclase during reoxygenation with urodilatin or cell-permeable cGMP analogues results in PKG-dependent phosphorylation of phospholamban, enhanced Ca2+ uptake by the SR, accelerated normalisation of cytosolic Ca2+ concentration, attenuated Ca2+ oscillations, and reduced probability of hypercontracture [40].

A second mechanism by which cGMP could prevent reperfusion-induced hypercontracture is by attenuating the sensitivity of the myocyte contractile system to Ca2+ [41]. PKG-mediated Ca2+ desensitisation could be related to direct phosphorylation of troponin I (TnI) and/or to direct or indirect effects of PKG on myosin light chain phosphatase (MLCP). Different observations suggest that PKG may phosphorylate the GTPase RhoA, reducing RhoA-dependent Ca2+ sensitisation of the myofilaments [42,43].

3.2 Attenuated contracture-dependent sarcolemmal rupture – role of calpains
Evaluation of calpain activation by different methods, including in vitro assay of its proteolytic activity with synthetic peptides, Western blot analysis of protein fragments resulting from hydrolysis of known calpain substrates, and quantification of its translocation to the membrane, has demonstrated that IP attenuates calpain activation occurring during reperfusion [13,44,45]. Moreover, calpain inhibitors partially reproduce the protective effects of IP, and treatments aimed to mimic or blunt the effects of IP respectively attenuate and normalise calpain activation secondary to ischemia–reperfusion [13]. Armstrong et al. observed that IP delays the onset of sarcolemmal fragility [46], and we recently have shown that IP prevents calpain activation, that this attenuates {alpha}-fodrin hydrolysis and the associated increase in fragility of cell membranes, and that calpain contributes through this mechanism to the protection induced by IP [13].

The kinetics of Na+ recovery during reperfusion determines the influx of Ca2+ through the reverse mode of NCX and, consequently, the degree of injury [47]. It has been suggested that IP accelerates Na+ kinetics during early reperfusion by preserving Na+/K+-ATPase function, and that this effect contributes to the protection afforded by IP [48–50]. We have recently shown that the effect of IP on the kinetics of Na+ recovery during reperfusion is mediated by attenuated calpain activation [13], resulting in attenuated displacement of Na+/K+-ATPase from the membrane–cytoskeleton complex, preserved Na+-pump activity during initial reperfusion, and attenuated Ca2+ influx through reverse-mode NCX (submitted data).

It has been proposed that attenuation of calpain activation can also protect the reperfused cardiomyocytes against mitochondria-driven cell death. Activated calpain, by cleaving Bid into an active form, has been described to induce the release of cytochrome c and other proapoptotic factors [44], and calpain inhibitors but not caspase inhibitors appear to preserve Bid cleavage and reduce cell death [51]. A more recent study in forebrain and liver mitochondria [52] proposes that calpain-activated Bid induces Bak oligomerisation and mitochondrial outer membrane permeabilisation, granting calpain access to the intermembrane space and apoptosis-inducing factor (AIF) cleavage and release. These results indicate that calpain activation could initiate apoptosis by a pathway independent of caspases, and provides an additional mechanism for the protective effect of calpain inhibition in IP.

It has been proposed that IP attenuates calpain activation as a consequence of reduced Ca2+ entry during ischemia [44], but attenuation of the Ca2+ rise during index ischemia in IP hearts is still matter of debate. An alternative explanation for the inhibition of calpain arises from studies suggesting that calpains may be regulated by phosphorylation. Freshly purified calpains are highly phosphorylated [53] and contain several conserved consensus sites for protein kinase A (PKA) phosphorylation [54], and it has been suggested that phosphorylation at serine 369 by PKA maintains calpain in an inactive state [55]. There is evidence that preconditioning ischemia results in transient PKA activation, and that abolition of this activation blunts the protective effects of IP while transient PKA stimulation mimics them [13,56,57]. Whether PKA exerts its effect on calpain activity by direct phosphorylation of the protease or by the action of other proteins related to the pathway triggered by PKA activation remains to be elucidated.

3.3 Attenuated cell-to-cell propagation of necrosis and gap junction (GJ)-independent connexin43 (Cx43)-mediated effects
3.3.1 Gap junctions and hemichannels
GJ-mediated intercellular communication allows cell-to-cell propagation of hypercontracture and cell death during myocardial reperfusion [58,59]. This propagation appears to be mediated by passage of Na+ from ruptured cells to adjacent ones and subsequent reverse-mode NCX and Ca2+ overload in the adjacent cells [60]. Cell-to-cell propagation is halted when the cell receiving Na+ influx is either able to extrude it efficiently through Na+/K+-ATPase or can withstand excessive contractile activity without suffering sarcolemmal rupture [61], two aspects probably depending on the degree of previous Ca2+ overload and calpain activation (see above). GJ-mediated spreading of necrosis appears to contribute to final infarct size in hearts, and also to contribute to increased cell death in other tissues and organs subjected to different insults, including ischemia [14,62].

Because GJ were known to contribute to cell death and the end-effectors of preconditioning protection against cell death were not known, and because both preconditioning and GJ-mediated communication are tightly regulated by protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs) [63,64], it was speculated that Cx43 GJ could be end-effectors of preconditioning [65]. The initial hypothesis was that IP slows down normalisation of GJ-mediated communication during initial reperfusion and would thus have a protective effect similar to that of the administration of GJ uncouplers during initial reperfusion [59]. In fact, there is ample evidence that IP modifies the phosphorylation state of Cx43 and slows its dephosphorylation during sustained ischemia [66–68]. However, the results of studies examining the potential role of delayed GJ-mediated communication during initial reperfusion in preconditioning cardioprotection are inconclusive and contradictory. A study has described reduced cell-to-cell chemical coupling in intact, preconditioned, reperfused myocardium as assessed by analysis of GJ-permeable dye transfer, and the authors ascribe this effect to PKC{varepsilon}-mediated Cx43 phosphorylation [67]. Other studies have failed to detect any differences in the rate of recovery of electrical coupling, as assessed by the analysis of tissue electrical impedance [59], a method of limited value in the assessment of GJ permeability and chemical coupling. Thus, available evidence does not yield an explanation for the protective effect of IP in terms of delayed recovery of GJ communication and limited spread of necrosis.

Evidence from electrophysiological and dye uptake studies on the existence of single hemichannels has been described in several cell types, including isolated adult rabbit cardiomyocytes [69]. It is generally believed that opening of hemichannels would rapidly dissipate transmembrane gradients and would thus be incompatible with cell life. However, recent evidence suggests that hemichannels may open under certain circumstances, including hyperosmolarity, metabolic inhibition, positive voltages, changes in protein phosphorylation, or low extracellular Ca2+ concentrations [69–72], and may participate in a number of cellular processes such as volume regulation [73] and the release of metabolites like ATP [74], which are involved in paracrine signalling.

Because the opening of hemichannels can induce extreme cell swelling and cell death and can be modulated by preconditioning via changes in their phosphorylation status, they have been considered potential end-effectors of preconditioning protection. In fact, it has been shown that IP dramatically reduces reperfusion-induced myocardial oedema [75,76], and it has been proposed that changes in permeability through Cx43 hemichannels [73] could explain both reduced swelling and protection [67,77]. Based on experiments with mutant Cx43 able to form junctional plaques but not functional GJ channels, some authors have suggested that hemichannels mediate the cytoprotective effects of Cx43 overexpression against different type of insults, including ischemia [78]. However, it is extremely difficult or impossible to demonstrate with the tools presently available that any transmembrane molecular flux occurs through hemichannels [79]. The role of hemichannel opening in the genesis of ischemic injury and in the protective effect of IP remains to be elucidated [69,77].

In addition to their potential role as end-effectors, Cx43 hemichannels have been suggested to be involved in preconditioning signalling through transient opening or conformational changes connected to Scr-ERK [80], an aspect out of the scope of the present review.

3.3.2 GJ-independent roles of Cx43 in preconditioning protection
While experimental proof of the involvement of Cx43 GJ or hemichannels as end-effectors of preconditioning protection remains elusive, very solid evidence of the involvement of Cx43 in preconditioning has been recently obtained. It has been shown that the effects of preconditioning on myocardial Cx43 content and phosphorylation status are not a mere consequence of the protection against cell injury during ischemic–reperfusion, but could be observed in the absence of prolonged ischemia [81]. The most conclusive evidence for a role of Cx43 in IP has been provided by studies showing that Cx43 deficiency in Cx43+/ – mice completely abolished preconditioning protection [82,83]. Interestingly, because this effect could be observed in cardiomyocytes isolated from these mouse hearts, the abolition of protection by Cx43 deficiency cannot be explained by changes in cell–cell communication [84]. Potential GJ-independent effects of Cx43 include, in addition to the elusive effects on potential hemichannels (see above), the participation of this protein as an element in signal transduction cascades. Proteomic studies have demonstrated that Cx43 participates in the formation of signalling complexes that may play a critical role in the cellular response to PCK{varepsilon} activation [85].

A breakthrough in the elucidation of the involvement of Cx43 in preconditioning may have been recently achieved with the description of the presence of Cx43 in the mitochondria of cardiomyocytes from mice, rats, pigs and humans [86]. It has also been demonstrated that brief preconditioning ischemia is rapidly followed by an increase in the mitochondrial localisation of Cx43, and recent studies show that Cx43 is localised at the inner mitochondrial membrane where it is translocated through the translocase of the outer mitochondrial membrane and the translocase of the inner mitochondrial membrane (TOM–TIM) general protein import system in a heat shock protein 90 (HSP90)-dependent way [87]. Deficiency of Cx43 at the inner mitochondrial membrane in Cx43+/ – mice abolishes diazoxide (DZX)-induced preconditioning, apparently by attenuating reactive oxygen species (ROS) generation and signalling [88]. The impairment of ROS generation in Cx43+/ – mice was selective for DZX and was not observed with other ROS generators such as menadione or valionomycin. Moreover, inhibition of Cx43 translocation to the inner mitochondrial membrane with geldanamycin also abolished DZX-induced protection [87]. These results suggest that that the mitochondrial localisation of Cx43 participates in ATP-dependent K+ channel-mediated signalling. Nevertheless, it must remembered here that the molecular identity of mitochondrial ATP-dependent K+ (mitoKATP) channels, the mechanisms by which their opening leads to prevention of cell death, or the potential interaction with MPTP have not yet been resolved. Further studies are clearly necessary to elucidate the exact mechanism by which mitochondrial Cx43 participates in protection against cell death and whether it does it as a mediator or as an end-effector.

3.4 Attenuated MPTP opening
Although the studies suggesting a role of inhibition of MPTP in the genesis of the protective effect of IP are less than 4 years old, the idea that MPTP is the fundamental end-effector of preconditioning protection has rapidly gained weight and seems to be widely accepted. The evidence described mainly in 5 recent original contributions has been extensively reviewed (see Gateau-Roesch et al., this issue (pages 264–273)). Both original and review articles concur in considering the evidence of the end-effector role of MPTP as solid. It would be redundant to review that evidence here; however, we believe that it is important to put it in context with other potential end-effectors.

3.4.1 Evidence of an end-effector role of MPTP in IP
The first study suggesting a role for MPTP in preconditioning protection observed that CsA had a protective effect similar to that of ischemic or pharmacological (with DZX) preconditioning in isolated rat hearts subjected to ischemia and reperfusion, and that the MPTP opener atractyloside abolished preconditioning protection. These authors also described an increased resistance to Ca2+-induced calcein release in isolated mitochondria treated with DZX [89]. This circumstantial evidence did not demonstrate that attenuated MPTP was the cause of protection, and the study provided no direct proof that MPTP was actually attenuated in preconditioned hearts. The authors concluded that "one interpretation of these data is that IP and mitoKATP channel activation may protect the myocardium by inhibition of MPTP opening at reperfusion" [89].

The first and thus far only study providing direct evidence of attenuated MPTP opening in preconditioned intact heart used the [3H]DOG mitochondrial entrapment method in perfused rat hearts [17]. The protective effect of CsA and SanglipherinD against MPTP opening occurring during reperfusion was also tested and, interestingly, was found to be less intense than that provided by IP. However, in this study, MPTP opening occurred more readily in mitochondria isolated from preconditioned hearts. The authors concluded that the protective effect of preconditioning on MPTP opening is probably indirect, through attenuated Ca2+ overload or ROS production. On the other hand, an important feature of the [3H]DOG entrapment method is that in cells in which membranes have been massively disrupted and [3H]DOG has been lost from the cytosol, MPTP opening does not result in mitochondrial [3H]DOG loading [2,17]. The method is thus blind to MPTP opening occurring as a consequence of sarcolemmal rupture. This feature, which is an advantage when trying to establish the role of MPTP opening as a cause of cell death, may be a limitation when the contribution of reduced MPTP opening to preconditioning cardioprotection is to be evaluated. In fact, in isolated rat hearts subjected to transient ischemia, the main effect of preconditioning protection is to reduce necrotic cell death resulting in massive enzyme release during the very first minutes of reperfusion, a type of cell death in which the role of MPTP opening cannot be properly evaluated by the [3H]DOG entrapment method. In our opinion, although this study provides the best available direct evidence of attenuated MPTP opening in preconditioning, it does not objectively demonstrate that this effect is responsible for a prominent fraction of the reduction in cell death afforded by IP.

None of the other studies demonstrates attenuated MPTP opening in intact preconditioned hearts [90–93]. One of these studies analysed the tolerance to Ca2+ in mitochondria isolated from preconditioned hearts [90]. However, it remains to be established whether the changes observed in mitochondrial performance are a cause or consequence of preconditioning protection. In addition, it remains to be established that tolerance to Ca2+ concentrations well above those attainable during ischemia–reperfusion truly correlate with reduced MPTP opening during reperfusion in intact myocardium, a situation during which other triggers seem to be more important than Ca2 [91]. Other studies demonstrated an increased tolerance to laser-induced MPTP opening in isolated cells preconditioned by hypoxia or DZX [92,93], without demonstrating a causative role of attenuated MPTP opening in preconditioning protection during real reperfusion. Finally, the increase in mitochondrial tolerance to ROS in pharmacologically preconditioned myocytes submitted to anoxia-reoxygenation and the potential mechanisms involved were investigated recently in an exhaustive and elegant study [93]. This study highlighted the role of ROS in MPTP opening during reoxygenation and the importance of mitochondrial depolarisation in cell death and provided solid proof of the key role of glycogen synthase kinase-3β (GSK3β) as modulator of MPTP. According to this study, the inhibitory effect of GSK3β on the MPTP complex limits MPTP opening induction and represents a general mechanism of cardioprotection (see later), although, again, it did not provide direct evidence of the importance of MPTP as an effector of preconditioning protection. It must be emphasised that in this study, anoxia-reoxygenation was performed at pH 7.4. Considering the marked inhibitory effect of acidosis on MPTP opening, this may be an important difference with respect to ischemia–reperfusion, which is associated with severe acidosis starting 5 min after the onset of ischemia to a few minutes after reperfusion.

In conclusion, even admitting that inhibition of MPTP may be an important end-effector of preconditioning, the mechanism responsible for this inhibition has not been resolved yet. It has been proposed that attenuated Ca2+ overload, improved ATP synthesis, and reduced ROS generation could contribute [2], but no direct proof of it has been so far provided. Recent studies suggest that modulation of GSK3β may play an important role.

3.4.2 The mechanism of MPTP inhibition in IP
It has been suggested that IP may indirectly inhibit MPTP during reperfusion by attenuating the stimuli that trigger its opening [2,17,94]. In this regard, great emphasis has been placed on the potential role of changes secondary to the opening of mitoKATP channels. A wealth of studies support the involvement of these channels, whose molecular identity remains unknown and whose very existence has been questioned [95], as mediators of preconditioning protection (recently reviewed in [96]). It has been suggested that opening of mitoKATP, and possibly other K+ channels, causes mitochondrial depolarisation and oedema, resulting in attenuated Ca2+ overload during ischemia and improved contact between the inner and outer membranes with favourable effects on adenine nucleotide depletion from the matrix and rapid restoration of ATP synthesis during subsequent reperfusion [94]. Beyond the controversy on the magnitude of mitochondrial depolarisation and Ca2+ overload inducible by mitoKATP channel opening, the protective effect of this reduction has never been experimentally demonstrated. In fact, there is increasing evidence indicating that Ca2+ may not be a relevant trigger of MPTP during myocardial reperfusion [93,97]. We have failed to detect any influence of Ca2+ overload occurring in isolated mitochondria during simulated ischemia (NaCN at pH 6.4) or anoxia on Ca2+-induced MPTP opening during subsequent re-energisation (submitted data). However, mitochondrial K+ influx through activated mitoKATP channels could have protective effects during reperfusion, since it should prevent matrix shrinkage associated with hyperpolarisation, thus attenuating Ca2+ influx and ROS generation during re-energisation [96]. In summary, different theories have been put forward to explain attenuated MPTP opening at the time of reperfusion in preconditioned myocardium as a consequence of reduced mitochondrial Ca2+ overload, ATP depletion, and ROS production secondary to mitoKATP channel activation, but experimental evidence supporting them is scant thus far. During recent years, it has been proposed that preconditioning could increase the sensitivity of MPTP to these triggers, and it has been suggested that GSK3β could play a key role in this regard.

Since GSK3 was initially described as a putative effector of the phosphatidylinositol 3-kinase (PI3K)–Akt protective pathway [98] in cultured cells, many studies have highlighted the importance of the β isoform of this enzyme for cell survival. Inhibition of GSK3β activity through phosphorylation is protective against several insults in different organs and tissues, including the heart [99,100]. It has been described that infarct size reduction afforded by IP is a GSK3-dependent process; blockade of GSK3β phosphorylation through PI3K inhibition with wortmannin ablated the protective effects of IP. Moreover, lithium or SB216763 (both GSK3β inhibitors) pre-treatment (before ischemia) mimicked the protective effects of IP in a perfused rat heart model [101], and subsequent work has extended this concept to other protective pathways, including opioids and adrenomedullin [102–104]. The broad spectrum of protective pathways converging on GSK3β has led to its consideration as an important end-effector of cytoprotection and, in particular, of preconditioning. A recent study provides evidence that in a cardiomyocyte model of hypoxia-reoxygenation, hypoxic and pharmacological preconditioning, triggered by a broad variety of drugs and mediated by a variety of signalling pathways including PKA, Akt, PKC, or mTOR, is due to GSK3β-dependent modulation of MPTP [93]. This makes GSK3β an attractive therapeutic target for which several pharmacological inhibitors are already available for human use [105]. The therapeutic potential is increased by the observation that GSK3β inhibition upon reperfusion is sufficient to elicit protection [102].

It has been suggested that increased NO availability in preconditioned myocardium during reperfusion could have an inhibitory effect on MPTP opening, but information available on this effect is inconsistent. In some studies, MPTP opening was observed in mitochondria exposed to S-nitrosothiols or concentrations of NO donors in the micromolar order, possibly in relation to nitrosative stress or by S-nitrosation of mitochondrial proteins [106], but other studies have described that NO donors prevent Ca2+-induced MPTP opening through a cGMP- and PKG-mediated effect [107]. MPTP modulation by the NO/cGMP pathway has been explained by changes in the concentration of Bcl-2 [108] or in the depolarisation of the mitochondrial membrane [109]. The importance of NO on the regulation of mitochondrial function has been recently reviewed [110].

Finally, other modulators of MPTP have been proposed to participate in preconditioning protection, but only based on indirect, circumstantial evidence. For example, based on the observations that pharmacological inhibition of the mitochondrial Ca2+ uniporter during reperfusion mimics IP protection and that uniporter openers abolish it, it has been proposed that IP reduces MPTP opening by inhibiting this mitochondrial Ca2+ channel [111].


   4. Conclusion
Top
 Abstract
 1. Introduction
 2. The effectors of...
 3. The effectors of...
 4. Conclusion
References
 
Despite the huge research effort dedicated to elucidate the mechanisms of the protection against cell death afforded by IP, our understanding of the end-effectors involved is very limited. There is evidence that IP attenuates hypercontracture-mediated cell death, at least in part through attenuated calpain activation during reperfusion, leading to preserved cytoskeletal integrity and improved Na+/K+-ATPase recovery and Ca2+ homeostasis. Various studies suggest that IP can reduce MPTP opening during reperfusion and limit infarct size through this mechanism. However, although generally accepted as a most important end-effector of protection, the contribution of MPTP to the infarct size reduction induced by IP in the intact heart needs to be established. This will be a difficult task due to the unknown genetic and molecular identity of the MPTP, the technical difficulties to monitor MPTP opening in intact myocardium during reperfusion, and the lack of potent and highly specific pharmacological modulators of this channel.


   Acknowledgements
 
This study was partially supported by grants CICYT SAF 2002-0559 and FIS-RECAVA. Antonio Rodríguez-Sinovas (FIS 99/3142), Alberto Cabestrero and Luis Agulló are supported by grants from the Ministerio de Sanidad y Consumo.


   Notes
 
Time for primary review 30 days


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