Connexin 43 signalling and cardioprotection

Abstract

Connexin (Cx) 43 is the predominant protein forming gap junctions and non‐junctional hemichannels in ventricular myocardium. The Cx43 proteins are central to the cardioprotection afforded by ischaemic preconditioning (IP). The specific role of mitochondrial C×43 in protection by IP is reviewed.

In the present review, the structure and regulation of connexin (Cx) 43‐formed hemichannels and gap junctions and their role in cardioprotection by ischaemic preconditioning (IP) are discussed. Furthermore, the presence of Cx43 at other subcellular locations within cardiomyocytes—particularly in the mitochondria—is considered in the context of IP.

Structure, function and regulation of connexins

The proteins of the connexin gene family, comprising 20 members in the mouse and 21 members in the human genome,¹ are named according to their molecular weights. Most cells or tissues express more than one connexin—for example, Cx40, Cx43 and Cx45 in the adult myocardium. Cx40 is expressed mainly in the atria and Cx45 in the conduction system, whereas Cx43 is the predominant connexin in the ventricles (for review see van Veen et al²).

Cx43, like all other connexins, contains four transmembrane domains, two extracellular and one intracellular loop, and the amino and carboxy termini, which are located in the cytosol. The connexins differ in the length of their cytosolic carboxy terminus, which is characterised by the presence of several phosphorylation sites (except for Cx26, which is unphosphorylated³).

Cx43 is predominantly located in the sarcolemma, where six connexins assemble into a so‐called connexon or hemichannel. Two opposing connexons, one from each adjacent cell, form a pore, which is central to electrical cell coupling. Clusters of such pores form a gap junction. Gap junctions are not selective for specific ions⁴ and are permeable for molecules with a molecular weight up to 1000 Da. The flux of molecules through gap junctions is determined by the chemical and electrical gradient between two connected cells. Furthermore, the transfer of molecules between connected cells is regulated by the assembly and degradation of gap junctions,⁵ as well as by their open probability.⁶ Most of the ventricular Cx43‐formed connexons are located in the terminal intercalated disks and some in the lateral sides of cardiomyocytes.

Non‐junctional hemichannels have been shown to contribute to volume regulation,⁷ the release of ATP and NAD⁺ from the cytosol,⁸ and the activation of cell survival pathways.⁹ Under resting conditions, hemichannels are predominantly in a closed state and their gating is regulated, among other factors, by the phosphorylation status of Cx43 (for review see Saez et al¹⁰). The number of putative phosphorylation sites of Cx43 is species dependent: 24 phosphorylation sites in mice and 19 in humans are predicted.² Cx43 is a target protein of several kinases, among them protein kinase A, protein kinase C (PKC), protein kinase G, protein tyrosine kinases, mitogen‐activated protein kinases and casein kinase, but also of protein phosphatases (for review see Schulz and Heusch¹¹ and Lampe and Lau¹²). The phosphorylation of Cx43 at Ser368 by PKC induces hemichannels to close, whereas inhibition of PKC induces them to open.¹³,¹⁴

Analysis of protein–protein interactions shows that Cx43 is associated not only with protein kinases but also with a variety of other proteins. Among these interaction partners are adherens junction‐ and tight junction‐associated proteins such as β‐catenin or ZO‐1 and ZO‐2, cytoskeletal proteins such as actin and tubulin, and caveolin (for review see Wei et al¹⁵ and Giepmans¹⁶). Recently, connexin‐interacting protein 150—a protein with no conserved domains —was found to interact with Cx43,¹⁷ and connexin‐interacting protein 150 has been suggested to be involved in regulating the Cx43 content in the plasma membrane. The finding that Cx43 interacts with several proteins exerting different functions supports the idea that Cx43 not only acts as a channel‐forming protein, but is also involved in intracellular signalling.

Cx43 in ischaemia and IP

Under physiological conditions Cx43 is partially phosphorylated and remains phosphorylated during the first few minutes of ischaemia.¹⁸ Ischaemia affects the association of Cx43 with kinases such as c‐Src in astrocytes.¹⁹ Several studies have shown dephosphorylation of Cx43 with an increasing duration of myocardial ischaemia,¹⁸,²⁰,²¹,²² a process associated with the opening of Cx43‐formed hemichannels.²³,²⁴ Ischaemia also induces translocation of Cx43 from the plasma membrane to an intracellular pool.²⁰

IP, the reduction of infarct size by brief episodes of ischaemia and reperfusion preceding a period of sustained ischaemia/reperfusion, is known to affect the phosphorylation status of Cx43. In pig,¹⁸ rabbit,²² and rat hearts,²¹ IP attenuated the ischaemia‐induced dephosphorylation of Cx43 and the resulting electrical uncoupling.²¹ The preserved phosphorylation of Cx43 after IP not only may be caused by an enhanced association of Cx43 with kinases such as PKC and p38¹⁸ but may also be due to reduced co‐localisation with protein phosphatases.²⁵

A so‐called death factor may spread from cell to cell through Cx43‐formed gap junctions at the time of reperfusion after sustained ischaemia.²⁶,²⁷ Uncoupling of gap junctions by heptanol may limit the spread of this factor—potentially sodium—and therefore the spatial progression of cell death.²⁸,²⁹,³⁰,³¹ On the other hand, pretreatment with heptanol, which dissolves Cx43 from membranes, abolishes IP cardioprotection,³² suggesting that Cx43 is central to the signal transduction cascade of IP.

Further evidence for the involvement of Cx43 in IP cardioprotection comes from experiments on heterozygous Cx43‐deficient mice, where IP cardioprotection is lost.³³,³⁴ Cx43‐formed gap junctions are not required for IP protection, as loss of IP protection after simulated ischaemia/reperfusion is also seen in isolated cardiomyocytes of Cx43‐deficient mice.³⁵ These findings imply a role of Cx43 in preconditioning that is independent of cell to cell communication.

Mitochondrial Cx43 in cardioprotection

Although Cx43 is predominantly located in the sarcolemma, the protein is also present in other subcellular structures. The cytosolic carboxy terminus of rat Cx43 (residues 243–382) is associated with the nucleus of cardiomyocytes and HeLa cells, where the stable expression of carboxy terminal Cx43 inhibits cell proliferation.³⁶ Furthermore, Cx43 is located in mitochondria of human umbilical vein endothelial cells, and the mitochondrial Cx43 content is increased in response to homocysteine‐induced cellular stress.³⁷ We have recently presented evidence for the presence of Cx43 in mitochondria from mouse, rat, pig and human left ventricular myocardium obtained from fluorescence‐activated cell sorting and western blot analyses, as well as confocal and immunoelectron microscopy.³⁸ In conditional Cx43 knockout mice the Cx43 content is reduced both in total ventricular extracts and in isolated mitochondria (fig 1). The location of Cx43 in mitochondria is of particular interest, because mitochondria are important signalling elements of IP.³⁹,⁴⁰,⁴¹,⁴² The mitochondrial content of Cx43 is increased very rapidly by IP (two 5 min cycles of ischaemia and reperfusion) and the enhanced Cx43 protein level in the mitochondria is maintained for at least 90 min in a pig model of IP.³⁸ The subcellular origin of Cx43, which is targeted for mitochondrial import, has not been elucidated. However, the fact that the Cx43 content of the intercalated disks is unaffected by IP¹⁸ makes it unlikely that Cx43 is transported from the sarcolemma to the mitochondria. It is also unclear whether Cx43 forms pore structures in the mitochondrial membrane as it does in the sarcolemma.

Figure 1 (A) Western blot analysis for connexin (Cx) 43 or as loading control for GAPDH on 30 μg ventricular protein extracts of Cx43^fl/fl, untreated Cx43^{Cre‐ER(T)/fl} or 4‐hydroxytamoxifen (4‐OHT) ‐treated Cx43^{Cre‐ER(T)/fl} mice. (B) Western blot analysis for Cx43 or for the mitochondrial marker protein ANT on 30 μg protein extracts of ventricular mitochondria isolated from Cx43^fl/fl, untreated Cx43^{Cre‐ER(T)/fl} or 4‐OHT‐treated Cx43^{Cre‐ER(T)/fl} mice. (C) Mitochondria isolated from the ventricles of Cx43^fl/fl, untreated Cx43^{Cre‐ER(T)/fl} or 4‐OHT‐treated Cx43^{Cre‐ER(T)/fl} mice were stained with antibodies against Cx43 (red) or the mitochondrial marker cytochrome c (green) and analysed by confocal laser scan microscopy. Merged image shows co‐localisation pixels in yellow. Reproduced with permission from Boengler et al.³⁸

Co‐immunoprecipitation studies have shown an interaction of Cx43 with Tom20, which is, with Tom5, 6, 7, 22, 40 and 70, part of the translocase of the outer membrane (TOM) protein complex and thereby of the general mitochondrial import machinery. Ischaemia decreases the mitochondrial protein level of Tom20, which functions as a presequence receptor for proteins to be imported into the mitochondria. However, in mitochondria of preconditioned pig myocardium, the Tom20 protein level is preserved.⁴³ The sustained Tom20 protein level may reflect overall preservation of the TOM complex during ischaemia after IP. Preservation of the TOM complex may enhance the translocation of Cx43 to cardiomyocyte mitochondria and thereby contribute to the increased mitochondrial Cx43 protein level after IP.

Functional role of mitochondrial Cx43

Reactive oxygen species (ROS) are produced in part by uncoupling of the oxidative phosphorylation during ischaemia/reperfusion and contribute to cardiomyocyte damage (for review see Ferrari⁴⁴). Whereas excessive ROS formation is detrimental to cardiomyocytes, small amounts of ROS trigger IP cardioprotection. ROS are a central step in the IP signal transduction cascade leading to activation of PKC⁴⁵ or to phosphorylation of p38 mitogen‐activated protein kinase and nuclear translocation of nuclear factor κB.⁴⁶ ROS are also second messengers in pharmacological preconditioning with diazoxide.⁴⁷,⁴⁸ Accordingly, we recently showed that diazoxide impaired ROS generation in heterozygous Cx43‐deficient cardiomyocytes compared with wild‐type cardiomyocytes. Loss of ROS formation in Cx43‐deficient cardiomyocytes was specific for diazoxide, as ROS formation did not change in response to the potassium ionophore valinomycin and to menadione, which non‐specifically induces ROS formation (fig 2). Consequently, diazoxide reduced infarct size in wild‐type but not in heterozygous Cx43‐deficient mice. In contrast, menadione reduced infarct size regardless of the genotype.⁴⁹ Therefore, heterozygous Cx43‐deficient cardiomyocytes have a specific functional deficit in ROS formation in response to diazoxide and accordingly have less protection. However, the function of Cx43 in mitochondria and its role in IP cardioprotection remains to be elucidated in more detail.

Figure 2 (A) Increase in MitotrackerRed (MTR) fluorescence reflecting reactive oxygen species (ROS) formation in response to diazoxide (200 µmol/l) above vehicle control was attenuated in cardiomyocytes from connexin (Cx) 43^+/– (n  =  10 mice) v wild type (WT) (n  =  12 mice). (B) ROS formation triggered by menadione (2 µmol/l) was not different between Cx43^+/– (n  =  5 mice) and WT (n  =  5 mice). (C) ROS formation by valinomycin (10 nmol/l) was also not different between Cx43^+/– (n  =  5 mice) and WT (n  =  5 mice). *p < 0.05 v WT. Reproduced with permission from Heinzel et al.⁴⁹

Conclusion

In all species analysed so far IP has been shown to be cardioprotective.⁵⁰ There is now evidence that IP‐induced and diazoxide‐induced protection depend on Cx43 but not on the presence of gap junctions. Instead, the location of Cx43 in mitochondria, which are organelles central to IP signal transduction, and the involvement of ROS formation suggest that Cx43 has a role in IP cardioprotection beyond its channel‐forming properties in the sarcolemma.

Abbreviations

Cx - connexin

IP - ischaemic preconditioning

PKC - protein kinase C

ROS - reactive oxygen species

TOM - translocase of the outer membrane