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Cardiovascular Research Advance Access originally published online on November 9, 2007
Cardiovascular Research 2008 77(2):325-333; doi:10.1093/cvr/cvm062
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007. For permissions please email: journals.permissions@oxfordjournals.org

Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia–reperfusion injury

Marisol Ruiz-Meana1, Antonio Rodríguez-Sinovas1, Alberto Cabestrero1, Kerstin Boengler2, Gerd Heusch2 and David Garcia-Dorado1,*

1 Servicio de Cardiología, Institut de Recerca, Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain
2 Institut für Pathophysiologie, Universitätsklinikum Essen, Germany

* Corresponding author. Tel: +34 93 489 4038; fax: +34 93 489 4032. E-mail address: dgdorado{at}ir.vhebron.net

Received 2 October 2007; revised 29 October 2007; accepted 30 October 2007

Time for primary review: 7 days


   Abstract
Top
 Abstract
1. Connexin43 as an...
 2. Synchronization of ischaemic...
 3. Cx43 is involved...
 4. Mitochondrial Cx43
 5. Clinical relevance
 6. Biological significance
 7. Conclusion
 Funding
 References
 
Connexins are transmembrane proteins whose best known function is to form gap junction channels. Ventricular cardiomyocytes express the connexin isoform Cx43 and are rich in gap junctions essential for the normal propagation of the action potential. In addition to this physiological role, cardiomyocyte gap junctions contribute to the pathophysiology of ischaemia–reperfusion injury, mainly by synchronizing the onset of rigour contracture during ischaemia and cell-to-cell propagation of hypercontracture during reperfusion. More recently, it has been recognized that Cx43 plays a role in protection during ischaemic and pharmacological preconditioning and that this role is independent from gap junction-mediated communication. It was demonstrated that Cx43 is localized in cardiomyocyte mitochondria, at least in part in the inner mitochondrial membrane, where it is imported by the general mitochondrial membrane translocase system. Interfering with this import system or genetic ablation of Cx43 abolishes diazoxide-induced protection, indicating that mitochondrial Cx43 participates in the preconditioning cascade. The role of mitochondrial Cx43 in preconditioning appears to be related to reactive oxygen species signalling, but its molecular mechanisms have not been elucidated in detail. The present article reviews available evidence on the localization of Cx43 in cardiomyocyte mitochondria, its role in protection by preconditioning, and the potential molecular mechanisms involved. These data may help to understand the pathophysiology of myocardial ischaemia–reperfusion and ischaemic preconditioning and to identify new strategies for cardioprotection, and they may be particularly relevant to situations such as aging in which total and mitochondrial Cx43 contents have been shown to be reduced.

KEYWORDS Myocardial infarction; Connexins; Cardioprotection; Mitochondria; Gap junctions; Ischaemia; Reperfusion

Connexins are transmembrane proteins able to polymerize into hexamers that form transmembrane channels. Hemichannels tend to gather in specialized areas of cell membranes and to dock to other hemichannels from an adjacent cell to form gap junction channels.1,2

Connexins form a large family of phylogenetically well-preserved proteins that are specifically or preferentially expressed in different cell types. All connexins contain four transmembrane domains connected by two cytosolic and two extracellular segments flanked by intracellular amino and carboxy terminal ends. The genes, structure, and cell-type expression patterns of the different connexins have been recently and extensively reviewed, as well as the functional properties, molecular interactions and regulation of the connexin hemichannels and gap junction channels according to their connexin composition, and will not be described here.1–4

Gap junctions were first observed about 40 years ago in cardiomyocytes, where they are very abundant. In adult myocardium, gap junctions localize almost exclusively at highly specialized membrane regions connecting and anchoring cardiomyocytes end-to-end (intercalated disks) where they play an essential role in the fast propagation of action potential necessary for coordinated contraction.5,6 The description of this physiological role of gap junctions and connexins was followed during the next decades by the identification of these structures in many other cell types and the recognition of their fundamental importance in many physiological and pathophysiological processes.7,8


   1. Connexin43 as an important player in the pathophysiology of myocardial ischaemia–reperfusion injury
Top
 Abstract
 1. Connexin43 as an...
2. Synchronization of ischaemic...
 3. Cx43 is involved...
 4. Mitochondrial Cx43
 5. Clinical relevance
 6. Biological significance
 7. Conclusion
 Funding
 References
 
Electrophysiological studies using different models and patch–clamp configurations and studies in papillary muscle preparations demonstrated more than two decades ago that acidosis, increased cytosolic concentration of Ca2+, amphipathic metabolites, and other changes occurring during ischaemia markedly reduced cell-to-cell electrical coupling.9–11 Detailed cable analysis demonstrated in ischaemic papillary muscle that the onset of rigour contracture and cytosolic [Ca2+] rise is immediately followed by a rapid increase in intercellular (gap junction-dependent) electrical resistance. It has been clearly shown that altered electrical coupling, together with cell depolarization and changes in action potential, contribute to the progressive reduction of impulse propagation velocity and eventual conduction blockade observed during ischaemia,9,12,13 and that the inhomogeneity of these changes in electrical conduction across ischaemic myocardial tissue plays a central role in the genesis of potentially lethal ventricular arrhythmias.9,13 Moreover, ischaemia induces rapid changes in the phosphorylation of connexin43 (Cx43), with overall dephosphorylation and degradation of gap junctions observable within hours.14,15 Altered gap junction distribution may contribute to the electrophysiological substrate for re-entrant ventricular arrhythmias occurring during subacute and chronic myocardial ischaemia.16–18 The important role of phosphorylation as a regulatory mechanism of gap junction-mediated intercellular communication is explained by the many phosphorylation sites of Cx43. These sites are targeted by different kinases and result in different changes in synthesis, trafficking, function, and degradation of Cx43 channels.19–21 Not only gap junction-mediated intercellular communication but also Cx43 hemichannels contribute to cell swelling during ischaemia.22


   2. Synchronization of ischaemic injury and propagation of cell death
Top
 Abstract
 1. Connexin43 as an...
 2. Synchronization of ischaemic...
3. Cx43 is involved...
 4. Mitochondrial Cx43
 5. Clinical relevance
 6. Biological significance
 7. Conclusion
 Funding
 References
 
In addition to their role in ischaemic arrhythmias, gap junction-mediated intercellular communication has been shown to play other important roles in the pathophysiology of myocardial ischaemia. It allows propagation of electrical impulse, but also, the large conductance of Cx43 gap junction in their open state allows for the passage of many biologically active and pathophysiologically relevant molecules, from ions to cyclic nucleotides and a vast array of messengers.23 This may result in synchronization and/or propagation of cytosolic derangements during ischaemia–reperfusion. In fact, detailed analysis of infarct geometry and computer simulation studies suggested that the fate of individual cardiomyocytes exposed to ischaemia–reperfusion was influenced by that of their neighbouring cells.24 Studies on end-to-end connected pairs of isolated cardiomyocytes, submitted to simulated ischaemia and reoxygenation and involving the use of gap junction-permeant fluorescent dyes, demonstrated that sarcolemmal rupture-induced hypercontracture propagates to the adjacent cardiomyocyte by a mechanism fully dependent on gap junction permeability.25 It was found that the passage of Na+ through gap junctions followed by Na+/Ca2+ exchange through the sarcolemmal exchanger NCE1 was responsible for this phenomenon,26 which contributed to the final extent of post-reperfusion necrosis; the prevention of this by different maneuvers limited infarct size in different models.25,27 An open question was the mechanism by which cell-to-cell propagation of hypercontracture was stopped before involving the whole myocardium. The critical factor seems to be the degree of calpain-dependent proteolysis: only if it is severe enough to produce significant sarcolemmal fragility28 and Na+ pump failure is Na+ influx through gap junctions expected to result in severe Na+ overload, secondary Na+/Ca2+ exchange, hypercontracture, and cell death. Gap junction-mediated propagation of cell injury was also observed in other tissues and cell types under different situations.29–33 It was also shown in several cell types, including cardiomyocytes, that under certain circumstances, gap junction-mediated intercellular communication could allow buffering of cytosolic alterations and prevent cell death rather than increase it.34–36

The pathophysiological role of gap junction-mediated intercellular communication during reperfusion was consistent with the presumed open status of gap junctions in reperfused myocardium in which cytosolic derangements, in particular elevated cytosolic Ca2+ and H+ concentrations, are rapidly corrected.37 On the contrary, the well-accepted notion that gap junctions are closed during ischaemia was not compatible with a role of gap junctions in the progression of ischaemic injury. However, studies in isolated cell pairs suggested that gap junction-mediated intercellular communication during the initial minutes of ischaemia allows synchronization of the onset of rigour. Moreover, studies of gap junction-permeant dye transfer in isolated cardiomyocyte pairs and in rat ventricular myocardium indicated the presence of residual gap junction-mediated intercellular communication even after rigour onset, [Ca2+] rise, and conduction blockade have occurred.38


   3. Cx43 is involved in ischaemic preconditioning
Top
 Abstract
 1. Connexin43 as an...
 2. Synchronization of ischaemic...
 3. Cx43 is involved...
4. Mitochondrial Cx43
 5. Clinical relevance
 6. Biological significance
 7. Conclusion
 Funding
 References
 
The large amount of research carried out during the last three decades on ischaemic preconditioning has provided much information on the triggers and mediators of protection by preconditioning, but it has been much less productive regarding the identification of the end-effectors of protection.39 This is not surprising considering the lack of understanding of the final effectors of cell death during ischaemia–reperfusion. Sarcolemmal rupture secondary to Ca2+-dependent hypercontracture and proteolysis of sarcolemmal cytoskeleton were initially proposed as the main mechanism of reperfusion-induced cell death in the form of necrosis.40 It was then proposed that caspase-dependent apoptosis plays a major role in cell death secondary to ischaemia–reperfusion. This hypothesis is difficult to reconcile with the fact that most of the cell death induced by transient ischaemia occurs during the first minutes of reperfusion and is accompanied by enzyme release that is indicative of cell rupture,25,27,28,41 a hallmark of necrosis. Recently, it has been suggested that mitochondrial permeability transition (MPT) occurring during reperfusion is the ultimate cause for necrotic cell death during reperfusion, presumably through dissipation of mitochondrial membrane potential and energetic collapse.42–44 However, the connection between MPT pore opening and sarcolemmal rupture has not been clearly established. One possibility is that MPT pore opening results in mitochondrial Ca2+ release, favouring hypercontracture.45

Because it was clear that the signal transduction connecting preconditioning stimuli with reduced cell death in response to ischaemia–reperfusion involved the action of different kinase systems, it was proposed that the end effector of protection should be a molecule regulated by changes in the phosphorylation status that could directly modulate cell death. Cx43 fulfilled these criteria,21 and it was suggested that delayed recovery of gap junction permeability during reperfusion could explain the protective effect of preconditioning by limiting cell death propagation. However, due to the high safety factor in gap junction-mediated intercellular communication, it is not clear that animals with a 50% reduction in Cx43 should have impaired recovery of intercellular communication during the initial minutes of reperfusion. In fact, results of studies measuring infarct size in these animals have shown variable results, and reduced infarcts as well as no effect have both been described.46,47 Studies with gap junction blockers suggested an involvement of Cx43 gap junctions in preconditioning,35 but the strongest evidence supporting the involvement of Cx43 in protection was the fact that preconditioning was not possible in Cx43-deficient mice (heterozygous Cx43 knockout; Cx43+/–).47,48

However, experimental evidence showed that the mechanism by which Cx43 was involved in preconditioning could not be explained by effects of preconditioning on gap junction-mediated intercellular communication. First, analysis of tissue electrical impedance indicated that preconditioning does not modify the rate of recovery of cell-to-cell electrical coupling during reperfusion.49 Secondly, Cx43 was shown to be also necessary to precondition isolated cardiomyocytes.50 These results suggested that Cx43 functions other than gap junction-mediated intercellular communication were involved in preconditioning protection.

Several studies have shown that connexins may influence cell death/survival independently of gap junction communication.51,52 These mechanisms are summarized in Figure 1. In isolated C6 glioma cells, N2A neuroblastoma cells, and HeLa cells, forced expression of Cx43 increased survival rate under different insults, including Ca2+ overload, oxidative stress, metabolic inhibition, tamoxifen exposure, and UV irradiation, even in non-confluent cultures or in the presence of gap junction uncouplers.52 The potential mechanisms responsible for these functions include unopposed connexin hemichannels in the plasma membrane and other localizations of connexin molecules within the cell.


Figure 1
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  		Figure 1 Scheme summarizing the potential roles of Cx43 in the pathophysiology of ischaemia–reperfusion. Solid lines indicate roles for which there is experimental evidence. Broken lines indicate phenomena for which available evidence has been obtained under conditions other than ischaemia–reperfusion. PK, protein kinases; Src, Src tyrosine kinase.

 
Due to the high, non-selective conductance and permeability of gap junction channels, it was assumed that unopposed hemichannels should be in a closed state, as their opening would result in depletion of cytoplasmic metabolites and cause cell death. However, recent evidence suggests that hemichannels may open under physiological and pathological circumstances in a highly regulated manner.53 Opening of connexin hemichannels under ischaemic conditions has been demonstrated in different cell types including cardiomyocytes,54 where it has been proposed to contribute to reperfusion injury.55 The mechanisms by which hemichannels at the plasma membrane may modulate cell death are multiple and have been recently reviewed.8 However, cell membrane hemichannels are not expected to open during brief ischaemic episodes, as those triggering protection, and thus not to result in cell swelling. Moreover, ischaemic preconditioning limits cell swelling caused by subsequent prolonged ischaemia and reperfusion.65


   4. Mitochondrial Cx43
Top
 Abstract
 1. Connexin43 as an...
 2. Synchronization of ischaemic...
 3. Cx43 is involved...
 4. Mitochondrial Cx43
5. Clinical relevance
 6. Biological significance
 7. Conclusion
 Funding
 References
 
Both the entire Cx43 protein and its free carboxy terminus are found in the nucleus,57,58 where they could exert modulatory effects on DNA synthesis,59 cell growth or cell differentiation,60 and free carboxy terminal fragments of Cx43 have been also described in the cytoplasm of cultured cells.61 However, the potential pathophysiological role of this localization of Cx43 remains unknown. We hypothesized that Cx43 could localize at mitochondrial membranes, and that this localization could be essential for ischaemic preconditioning. The hypothesis of the presence of Cx43 in cardiomyocyte mitochondria was based on the following facts: (1) mitochondria are essential in the protection afforded by ischaemic preconditioning; (2) mitochondria have a double membrane system resembling that in gap junctions; (3) mitochondrial function is dramatically dependent on mitochondrial volume, and changes in mitochondrial volume appear to play an important role in ischaemic preconditioning; and (4) Cx43 may interact with many proteins62 and is targeted by many different protein kinases responsible for preconditioning signalling.

The presence of Cx43 in cardiomyocyte mitochondria was initially demonstrated by an array of immunodetection-based approaches showing the colocalization of Cx43 with mitochondria or mitochondrial proteins,63 including flow cytometry, confocal co-localization, and immunoelectron microscopy with gold bead-labelled antibodies. The purity of mitochondrial preparations from mice, rat, pig and human hearts was checked by Western blotting against proteins of other cell compartments. The specificity of the anti-Cx43 antibody used was supported by the absence of labelling in hearts from treated Cx43Cre-ER(T)/fl mice in which Cx43 expression was largely reduced by treatment with 4-hydroxytamoxifen. Meanwhile, in addition to the two laboratories involved in the initial report, other groups have confirmed the presence of Cx43 in purified mitochondrial fractions.64,65 Moreover, quantitative Western blot analysis of mitochondrial preparations purified with FACS demonstrated a higher content of Cx43 in rat cardiomyocyte mitochondria isolated after preconditioning, and Western blotting revealed increased content of mitochondrial Cx43 in preconditioned pig myocardium,63 findings that were in good agreement with a role of mitochondrial Cx43 in ischaemic preconditioning.

None of the mitochondrial proteomes published thus far have included Cx43.56,66 However, the fact that Cx43 does not appear in previously reported 2D electrophoresis-based mitochondrial proteomes is not surprising, since it is well known that those proteomes are not exhaustive and are particularly likely to miss membrane proteins.67 In order to prove the presence of Cx43 in mitochondrial membranes by antibody-independent methods, we performed HPLC chromatography in tandem with linear ion trap mass spectrometry using a method particularly suitable for specific identification of peptides in complex samples by which we had previously identified Cx43 in membrane preparations.68 The results unambiguously demonstrated the presence of Cx43 in a preparation of mitochondrial membranes.

4.1 Localization and mechanism of import of mitochondrial Cx43
Establishing the exact localization of Cx43 within mitochondria is of fundamental importance in elucidating its potential (patho)physiological role. In a recent study, we used laser confocal microscopy to demonstrate Cx43 immunoreactivity in digitonin-treated mitochondria devoid of voltage-dependent anion channel (VDAC) but conserving cytochrome C and adenine nucleotide translocase (ANT) immunoreactivity. Subfractionation and Western blot analysis demonstrated that Cx43 is present almost exclusively in the inner membrane.69 Other authors, by using idoxanol gradient centrifugation on enriched mitochondrial fractions, have described the presence of Cx43 mainly at the outer mitochondrial membrane.64 The reasons for this discrepancy are not clear, but they may be related to differences in the purity of mitochondrial preparations and in the subfractionating methods.

The presence of Cx43 at the inner mitochondrial membrane leads to the question of the mechanism by which it is imported to this location. Available evidence indicates that Cx43 gets to the inner mitochondrial membrane by using the general translocase of the outer membrane (TOM)/translocase of the inner membrane (TIM) import system69 (Figure 2). This system involves binding of the target protein to a chaperone (HSP90/HSP70), presentation to specific parts of the TOM complex (Tom20), transport to the TIM complex, and release into the inner mitochondrial membrane; such transport has been recently described with great detail.70 Import of proteins through the TOM/TIM system can be inhibited by the HSP90 inhibitor geldanamycin, and this was the case for Cx43. Moreover, co-immunoprecipitation studies indicated the interaction of Cx43 with HSP90 and TOM20. It could be estimated that, under basal conditions, most Cx43 protein is found at the intercalated disks, whereas only 4% is localized in mitochondria and 8% in the cytosol.


Figure 2
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  		Figure 2 Transport of Cx43 to the inner mitochondrial membrane and other cellular localizations. Broken arrows indicate inconclusive or contradictory evidence. TOM, translocase of the outer mitochondrial membrane; TIM, translocase of the inner mitochondrial membrane.

 
The very rapid increase (within minutes) in mitochondrial Cx43 content in response to ischaemic preconditioning indicates that it is due to changes in the intracellular kinetics of Cx43 rather than to increased protein synthesis. Transport through the TOM/TIM general import system can be regulated in many different ways,70 and information on the particular mechanisms regulating the import of Cx43 through this system is not yet available. Mitochondrial Cx43 exists almost exclusively in the phosphorylated form,63,64,69 indicating that phosphorylation can play an important role in regulating mitochondrial Cx43 content. The degradation process of Cx43 is still largely unknown.

4.2 Mitochondrial Cx43 and protectionby ischaemic preconditioning
Different proteins have been shown to translocate to mitochondria in response to different stimuli, including ischaemia,71 and translocation of Cx43 to the mitochondria during ischaemic preconditioning does not prove its causative role in cardioprotection, since genetic manipulation of Cx43 can influence protection by mechanisms independent from mitochondrial Cx43. Thus, in order to prove that mitochondrial Cx43 plays a role in preconditioning, it is necessary to identify the mechanism involved and to analyse the effects of selective changes in mitochondrial Cx43 on this mechanism.

Since mitochondrial reactive oxygen species (ROS) generation appears to play a key role in preconditioning signalling, it was hypothesized that mitochondrial Cx43 modulates mitochondrial ROS generation in response to preconditioning stimuli. This hypothesis predicts that during ischaemic preconditioning ROS generation is impaired in Cx43-deficient (Cx43+/–) mice. This was confirmed in isolated Cx43-deficient cardiomyocytes in which exposure to diazoxide resulted in markedly attenuated ROS generation and did not confer protection against subsequent ischaemia–reperfusion.72 In contrast, unspecific production of ROS by menadione and K+ ionophores as well as the resulting protection were not altered in Cx43-deficient myocytes.72 These results were consistent with the hypothesis of the modulation of mitochondrial ROS generation by Cx43, but left the possibility open that localizations of Cx43 other than mitochondria were responsible for this effect.

Another proof of the involvement of mitochondrial Cx43 in diazoxide-induced cardioprotection was obtained in experiments in which the amount of Cx43 localized in mitochondria was selectively reduced (without altering total Cx43 cell content) by geldanamycin. In these studies, reduction of mitochondrial Cx43 by geldanamycin was associated with the abolition of the cardioprotective effect of diazoxide against cell death induced by ischaemia–reperfusion, as measured by triphenyltetrazolium chloride reaction, histology, and lactate dehydrogenase release,69 whereas geldanamycin alone had no effect on cell death, indicating that mitochondrial Cx43 is essential for diazoxide-induced protection.69

Treatment with geldanamycin completely prevented the increase in mitochondrial Cx43 content induced by ischaemic preconditioning in isolated rat hearts. However, geldanamycin-treated hearts were still protected by ischaemic preconditioning.69 Thus, it appears that translocation of Cx43 to mitochondria is not essential for cardioprotection. This observation, together with the fact that Cx43-deficient mice in which mitochondrial Cx43 content was markedly reduced to ~20% of control values72 could not be preconditioned,47,48 suggests the existence of a threshold in mitochondrial Cx43 content below which the protection by ischaemic preconditioning is lost. The reason why pharmacological preconditioning with diazoxide is abolished by milder reductions in mitochondrial Cx43 could be related to the existence of different redundant, and to some extent additive, signal transduction pathways involved in protection.73–75 ROS generation represents only one of these pathways,73,76–78 and its ablation, while it may abolish protection induced by a drug such as diazoxide whose protective effect specifically relies on the ROS pathway,79,80 may not be sufficient to abolish protection induced by ischaemic preconditioning. Of note, Cx43 is not prerequisite for the protection of ischaemic postconditioning.81 In any case, understanding the mechanism by which Cx43 is involved in protection by preconditioning requires elucidation of its molecular functions.

4.3 Molecular mechanisms
A straightforward approach to determine the functional role of mitochondrial Cx43 is to analyse what particular functions are modified in isolated mitochondria by an increase or a decrease in mitochondrial Cx43 content. The most important mitochondrial function is respiration, and preliminary data indicate that it is significantly influenced by mitochondrial Cx43 content. Mitochondria from Cx43-deficient mice, Cx43Cre-ER(T)/fl, show attenuated ADP-stimulated (state 3) respiration and a reduced respiratory control ratio as compared with their control littermates,82 which is solid evidence of a direct functional role of mitochondrial Cx43. However, the mechanism by which Cx43 may influence mitochondrial respiration remains unknown. Cx43 could interact with the different complexes of the respiratory chain in the inner mitochondrial membrane or indirectly modulate their function. Matrix volume changes in response to different stimuli modulate mitochondrial respiration, and recent data in genetically modified mice in which Cx43 is replaced by Cx32 indicate that the presence of Cx43 markedly influences mitochondrial volume regulation.83 Further studies will be necessary to reveal these mechanisms.

A fundamental question in the elucidation of mitochondrial Cx43 functions is whether or not it forms hemichannels. The different possibilities are illustrated in Figure 1. Due to its ability to interact with different molecules, including protein kinases,84 cytoskeletal elements (zonula occludens 1 among others),62,85,86 and aquaporins,87,88 Cx43 would have important (patho)physiological functions by modulating the function of other molecules, e.g. KATP channels, without forming connexons. On the other hand, the presence of Cx43 hemichannels in mitochondrial membranes is expected to have important physiological consequences. The potential localizations of Cx43 hemichannels and channels in mitochondrial membranes are summarized in Figure 3. If present in the internal membrane, Cx43 hemichannels should normally be closed because their large conductivity would otherwise dissipate mitochondrial membrane potential, interrupt ATP synthesis, and cause massive mitochondrial swelling in a way similar to that described for MPT.43 However, the potential harmful consequences of their opening do not rule out their presence in the inner mitochondrial membrane. The same reasoning could be applied to other potentially harmful channels, such as the MPT pore, the existence of which has been solidly demonstrated and the transient opening of which has been shown to be compatible with cell survival.43 This pore has even been proposed to be necessary in the triggering of ischaemic preconditioning.89 In a similar way, opening of Cx43 hemichannels in the plasma membrane may also have detrimental consequences (massive cell swelling, Na+ and Ca2+ overload, and depolarization), yet there is little doubt of their presence at that location.53,54,90 It is thus conceivable that Cx43 forms hemichannels at the inner mitochondrial membrane in which the opening probability is very low under normal, physiological conditions. This, in fact, seems to be the normal situation in unopposed hemichannels.53 Functions of mitochondrial Cx43 connexin hemichannels must depend then on small and transient increases of the probability of switching to either full opening or incomplete opening with low conductance (residual conductance state). In fact, the existence of a residual conductance state of about 15–28 pS for Cx43, as compared with a maximum conductance for the open state of 115 pS, is widely known.91 Its existence is based on the fact that each Cx43 hemichannel in a gap junction channel possesses two different gates: a fast gate that closes channels to the residual state and a slow gate that fully closes the channels. Although the exact regulation of this residual state is not known, it seems to depend, at least in part, on transjunctional voltages91 through a gating element located in the vestibule of the pore.92


Figure 3
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  		Figure 3 Possible localizations of Cx43 in mitochondrial membranes. The point of contact between two mitochondria is represented. Cx43 could form hemichannels (A through E) or not (F and G). Hemichannels could allow water and the flux of molecules across the outer mitochondrial membrane (A), the inner mitochondrial membrane (E) or both (D), or could connect the intermembrane spaces (B) or the matrix (C) of two adjacent mitochondria. In D and E, hemichannels remain closed under normal conditions. Signal transduction could occur in all cases via interaction of Cx43 with other signal transduction elements, and in A through E, through fluxes of water, ions, and different messengers. Possibilities E and F have been supported by different experimental observations (in the text).

 
The movements of ions, water, and metabolites across mitochondrial Cx43 hemichannels would be, as in the case of membrane hemichannels, very difficult to dissect from fluxes of these molecules currently attributed to other, better-known channels and pores. This makes it possible that some of these fluxes could be in fact occurring through mitochondrial Cx43 connexons, which at the same time poses a great difficulty in demonstrating and characterizing the putative functions of these channels. Figure 4 summarizes some potential mechanisms by which mitochondrial Cx43 could be involved in preconditioning protection.


Figure 4
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  		Figure 4 Potential mechanisms by which mitochondrial Cx43 could participate in ischaemic pharmacological (diazoxide) preconditioning. Monomeric Cx43 (in blue) could modulate mitochondrial K+ATP channels (in brown), but also the effects of diazoxide on the respiratory chain (in dark gray).103 Cx43 hemichannels could favor H+ and K+ leak resulting in protective mild uncoupling104 and swelling.105,106

 

   5. Clinical relevance
Top
 Abstract
 1. Connexin43 as an...
 2. Synchronization of ischaemic...
 3. Cx43 is involved...
 4. Mitochondrial Cx43
 5. Clinical relevance
6. Biological significance
 7. Conclusion
 Funding
 References
 
Elucidation of the role of mitochondrial Cx43 in cardioprotection will improve our understanding of the pathophysiology of ischaemia–reperfusion injury and could lead to the development of strategies to limit cell death secondary to transient myocardial ischaemia. Beyond that, the functional characterization of the localization of Cx43 in cardiomyocyte mitochondria could have implications in many clinical situations in which both mitochondrial alterations and changes in cardiomyocyte Cx43 distribution have been described, including heart failure93,94 and aging.95,96

Aging has been shown to be associated with a reduction of Cx43 content in different cell types of several species, including cardiomyocytes.97 Recent data have demonstrated a reduction in total and mitochondrial Cx43 content in cardiomyocytes from aged mice that is paralleled by a loss of protective effects of ischaemic preconditioning.95 Because protection is largely independent of gap junction-mediated intercellular communication, these data suggest that loss of mitochondrial Cx43 could contribute to the loss of protection.

Identification of new functions of Cx43 in mitochondria may be relevant to other conditions in other cell types. Neurons, astrocytes, endothelial cells, Kupffer cells, and vascular and gastrointestinal smooth muscle cells express Cx43, and it is conceivable that the mitochondrial localization of this protein could have a role in the modulation of cell death and dysfunction in different pathological conditions and in their modulation by aging.


   6. Biological significance
Top
 Abstract
 1. Connexin43 as an...
 2. Synchronization of ischaemic...
 3. Cx43 is involved...
 4. Mitochondrial Cx43
 5. Clinical relevance
 6. Biological significance
7. Conclusion
 Funding
 References
 
The studies reviewed here describe a new localization of Cx43 in cardiomyocyte mitochondria with potentially important (patho)physiological functions, but the elucidation of these functions, their molecular mechanisms, and their regulation will require many additional studies. One fundamental question that needs to be addressed in order to understand the biological significance is, for example, whether the mitochondrial localization of Cx43 is specific for cardiomyocytes or whether it is true for other cell types expressing Cx43. The scant available data are compatible with the latter.98

It must also be determined whether connexins other than Cx43 may also localize at mitochondria. Preliminary data from transgenic mice in which Cx43 is replaced by Cx32 indicate that the ability to translocate to mitochondrial membranes may be different for the different connexins.83

Mitochondria and connexins represent two key steps in the evolution of animal life. Mitochondria are the hallmark of eukaryotic cells.99 The symbiotic incorporation of a metabolically versatile alpha-protobacterium (future mitochondria) into a larger anaerobic cell, with internalization and gene transfer, gave rise to the eukaryotic cell and represented a huge leap in size and complexity that was the basis of for all future evolution.99 Gap junctions are the hallmark of multicellular animals.100,101 In contrast to plants, animal members from a particular species have a very similar shape and size, reflecting a precisely coordinated development, and as compared with plants, animals react and move faster, reflecting rapid and efficient intercellular coordination provided by gap junction-mediated intercellular communication.23 The importance of gap junction-mediated intercellular communication is stressed by the striking fact that evolution has produced two genetically unrelated families of molecules able to form intercellular hexameric channels: connexins (in chordata),100 and innexins and pannexins (in all animals, including chordata).101,102 Among connexins, Cx43 is expressed in many cell types. Some of these cell types preferentially or exclusively expressing Cx43, in particular cardiomyocytes and neurons, share a high respiratory rate and a large mitochondrial mass. A mitochondrial localization of Cx43 with potential effects on the regulation of mitochondrial volume, respiration, and ROS generation could be biologically meaningful.


   7. Conclusion
Top
 Abstract
 1. Connexin43 as an...
 2. Synchronization of ischaemic...
 3. Cx43 is involved...
 4. Mitochondrial Cx43
 5. Clinical relevance
 6. Biological significance
 7. Conclusion
Funding
 References
 
Available evidence demonstrates the presence of Cx43 in cardiomyocyte mitochondria and indicates that this localization plays an important role in the cardioprotection afforded by preconditioning. A great deal of research will be necessary to elucidate the molecular mechanisms involved, but the results may open new horizons in our view of the pathophysiology not only of myocardial ischaemia–reperfusion injury, but of other conditions in the cardiovascular system and beyond.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Connexin43 as an...
 2. Synchronization of ischaemic...
 3. Cx43 is involved...
 4. Mitochondrial Cx43
 5. Clinical relevance
 6. Biological significance
 7. Conclusion
 Funding
References
 
This study was partially supported by the Spanish Ministry of Health-RETICS (RECAVA), FIS-PI060996, and CICYT SAF2005-01758 (Spanish Ministry of Education). A.R.-S. is a recipient of a contract from the Generalitat de Catalunya (Programa d’Estabilitzacio d’investigadors, Departament de Salut, Direcció d’Estratègia i Coordinació). K.B. was supported by the German Research Foundation (Schu 843/7-1).


   References
Top
 Abstract
 1. Connexin43 as an...
 2. Synchronization of ischaemic...
 3. Cx43 is involved...
 4. Mitochondrial Cx43
 5. Clinical relevance
 6. Biological significance
 7. Conclusion
 Funding
 References
 

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