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Biochemical Society Transactions (2015) Volume 43, part 3
Gap junctions – guards of excitability Line Waring Stroemlund*1 , Christa Funch Jensen*, Klaus Qvortrup†, Mario Delmar‡ and Morten Schak Nielsen* *Department of Biomedical Sciences and The Danish National Research Foundation Centre for Cardiac Arrhythmia, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark †Department of Biomedical Sciences and Core Facility for Integrated Microscopy, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark ‡Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, U.S.A.
Biochemical Society Transactions
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Abstract Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although these structures have long been known, it is becoming increasingly clear that their components interact. This review describes the involvement of the ID in electrical disturbances of the heart and focuses on the role of the gap junctional protein connexin 43 (Cx43). Current evidence shows that Cx43 plays a crucial role in organizing microtubules at the intercalated disc and thereby regulating the trafficking of the cardiac sodium channel NaV 1.5 to the membrane.
The intercalated disc The efficient activation and contraction of the heart requires that cardiomyocytes are coupled both electrically and mechanically, which occurs in the intercalated disc (ID). The ID was first observed by light microscopy in 1866 by Eberth [1]. In 1954 Sjostrand and Andersson [2] imaged the ID at high resolution using electron microscopy (EM), demonstrating its complex nature. Figure 1 shows an overview and details of a rat ID imaged by EM. The ID is a highly organized structure (Figures 1 and 2), composed of both anchoring complexes (fascia adherens junctions and desmosomes) and sites of intercellular communication (gap junctions). The mechanical connections in adherens junctions and desmosomes both include proteins of the cadherin family. The cadherin family is a class of proteins that dock with cadherins from neighbouring cells in a calcium dependent manner. On the intracellular side they link to the contractile machinery and intermediate filaments, and thus ensure both efficient transfer of mechanical force from cell to cell and mechanical stability of the cells (for review see Kline and Mohler [3]). The adherens junctions consist of the transmembrane protein N-cadherin. The extracellular domain is responsible for the calcium dependent binding to cadherins in neighbouring cells, whereas the intracellular domain is linked to the actin cytoskeleton through a complex involving α-catenin, βcatenin and vinculin [3]. Desmosomes consist of desmocollins and desmogleins that bind to each other through the extracellular domain. On the intracellular side, they form complexes with desmoplakin,
Key words: Cx43, intercalated disc, microtubule, Nav1.5, trafficking. Abbreviations: ACM, arrhythmogenic cardiomyopathy; AnkG, ankyrin-G; Cx43, connexin 43; EB1, plus-end tracking protein; EM, electron microscopy; ID, intercalated disc; IK , potassium current; INa , sodium current; NaV 1.5, sodium channel; PKP2, plakophilin-2; ZO-1, zonula occludens protein-1. 1 To whom correspondence should be addressed (email [email protected]).
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plakophilin-2 and plakoglobin and anchors to the desmin based intermediate filaments [3]. Gap junctions are composed of intercellular channels that allow passage of small ions and molecules between neighbouring cells. The channels are formed by proteins of the connexin family, where connexin 43 (Cx43) is the dominant isoform in the working myocardium. Six connexins form a connexon and when two connoxons from neighbouring cells dock in the extracellular space, they form an intercellular pore [4]. Although the adherens junctions, desmosomes and gap junctions are discernible as separate structures in EM, mixing of proteins from the adherens junctions and desmosomes has been reported. These areas of mixed-type junctions are referred to as the area composita [5]. Likewise, Cx43 has been located outside the gap junctional structure [6,7].
Intercalated disc diseases Several pathologies are related to mutations in ID components and the functionality of the ID. Some of these are acquired diseases, such as infarction, and associated with disarray of ID components, a disarray believed to contribute to arrhythmogenesis [8]. Acquired diseases are often complex, making it difficult to establish the exact causality, but lessons taught from rare genetic diseases have helped improve our current understanding of ID function. Arrhythmogenic cardiomyopathy (ACM) is formerly referred to as arrhythmogenic right ventricular cardiomyopathy (ARVC). The disease is characterized by fatty or fibrofatty replacement predominantly in the right ventricle which can lead to ventricular arrhythmias, ventricular dysfunction and cause of sudden cardiac death [9]. ACM has previously been considered a desmosomal disease as mutations were primarily found in the components of the desmosome, including plakophilin-2 (PKP2 gene), Biochem. Soc. Trans. (2015) 43, 508–512; doi:10.1042/BST20150059
8th UK Gap Junction Meeting
Figure 1 Electron micrograph of intercalated disc between rat cardiomyocytes (A) Electron micrograph showing an overview image of an intercalated disk (ID) between two rat myocytes, illustrating its stair-like structure, with fascia adherens (FA) running perpendicular to the fibre orientation and gap junctions (GJ) running in parallel often followed by a desmosome (D). (B) Magnified detail image of fascia adherens (FA), desmosome (D) gap junctions (GJ), and perinexus (PN).
Figure 2 Schematic representation of the cardiac intercalated disc components Simplified model of the molecular composition of the cardiac intercalated disc, including components of the desmosome (D), fascia adherens (FA), the cardiac sodium channel NaV 1.5, gap junction (GJ) and the cytoskeleton.
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desmoglein-2 (DSG2 gene), desmocollin-2 (DSC2 gene), desmoplakin (DSP gene) and plakoglobin (the JUP gene) [10]. However, ACM may also be caused by mutations in proteins localized outside the desmosome. Van Hengel et al. [11] screened 76 ACM patients without mutations in desmosomal genes and found two mutations in the CTNNA3 gene encoding αT-catenin which is expressed in the adherens junctions. Since then, ACM causing mutations have been described in additional ID components [10]. ACM can therefore no longer be considered as a purely desmosomal disease but rather a disease of the ID. Furthermore, mutations in components of the mechanical junctions also affect structures involved in the propagation and initiation of the cardiac action potential. Saffitz and co-workers showed that plakoglobin mutations causing Naxos disease were associated with significant gap junction remodelling [12]. In a comprehensive study of 20 patients, Noorman et al. [13] showed that not only Cx43 but also NaV 1.5 were severely affected in a large fraction of ACM patients. These findings indicate that gap junction and sodium channel remodelling is likely a common trait in ACM. More recently, it was shown that mutations in structural ID proteins need not lead to ACM but may result in electrical abnormalities without gross anatomical alterations. Specifically, Cerrone et al. [14] showed that a missense mutation leading to loss of PKP-2 reduced NaV 1.5 expression at the ID and decreased sodium current (INa ), which explained the Brugada syndrome phenotype of patients carrying the mutation. All of these observations point to the fact that alterations in any ID protein are likely to affect the entire ID. This in turn raises the question of the exact mechanisms that govern the localization and function of key proteins such as Cx43 and NaV 1.5. The gap junctional protein Cx43 is known to function, not only in its capacity of forming intercellular pores, but also as hub binding a multitude of partners. Evidence now shows that Cx43 may be a major organizer of transport to the ID.
Cx43 and the microtubular network Giepmans et al. [15] were the first to show that the Cterminal tail of Cx43 interacts directly with tubulin, and that microtubules preferentially terminates in areas of cell to cell contact. This led the authors to suggest that gap junctions act as anchoring points for microtubules. Since then studies have confirmed and elaborated on the Cx43 interactions with the microtubular network. Lo and coworkers showed that cytoskeletal organization and cell migration was dependent on the presence of Cx43 and that the deletion of a tubulin-binding motif recapitulated the phenotype of cells lacking Cx43 [16]. Based on these studies it seems that Cx43 is a key regulator of microtubule organization. However, the exact nature of microtubule binding to the ID still remains uncertain. Some studies suggest that the microtubule is interacting directly with Cx43 via
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microtubule binding domains [15,16], while other studies suggest that that microtubule plus-end tracking protein EB1 interacts (possibly via a secondary interactions) with Cx43 [7], adherens junctions [17] or desmoplakin [18]. Irrespective of the nature of EB1 binding, Cx43 seems to play an important role in the final stabilization of microtubules at the membrane. The interaction between the microtubule and Cx43 potentially affects not only Cx43 trafficking, but also the trafficking of other ion channels. This notion is supported by Jansen et al. showing that knockout of Cx43 reduced the expression of NaV 1.5, reduced INa in mouse ventricular myocytes, and made hearts more prone to ventricular arrhythmias [19]. Desplantez et al. [20] made similar observations on atrial myocytes from mice with genetic ablation of Cx43 showing decreased INa and demonstrating that the interaction between Cx43 and NaV 1.5 is not restricted to ventricular myocytes. NaV 1.5 is the main cardiac voltage-gated Na + channel and is found preferentially at the ID but also to lesser extent in costameric and T-tubular locations. The trafficking basis for this distribution is still unclear but membrane expression of NaV 1.5 depends at least partially on both SAP97 [21] and ankyrin-G (AnkG) [22]. The in vivo importance of AnkG was recently demonstrated by Mohler and co-workers, who showed that knockout of AnkG reduced NaV 1.5 specifically at the ID [23]. These findings do, however, not resolve the pathway by which NaV 1.5 is trafficked to the membrane, but Casini et al. [24] showed that interfering with microtubule polymerization led to reduced surface expression. This leaves Cx43 as an interesting candidate for leading microtubules and thereby NaV 1.5 to the ID. This hypothesis was directly addressed by Agullo-Pascual et al. [7] using HL1 cells expressing either wild type Cx43, Cx43 lacking the microtubule binding domain or lacking Cx43 altogether. The study showed that removing Cx43 entirely or removing the microtubule binding domain both reduced INa to a similar extent. The exact location of Cx43 microtubule interaction is unknown, but since it is unlikely that NaV 1.5 is delivered inside the gap junction plaques, the interaction probably occurs outside the plaque itself. One such location is the perinexus at the transition between the gap- and adherens junction (see Figure 1B) where Cx43 and NaV 1.5 colocalize [6]. More recently it was demonstrated that Cx43 is also located throughout the adherence junctions, where they (due to membrane spacing) are unlikely to form intercellular channels but could serve the function of anchoring microtubules [7]. At least in the perinexus Cx43 interacts with its scaffolding protein zonula occludens protein-1 (ZO-1) through a PDZ binding domain located in the last five amino acids of the C-terminus [25]. The influence of Cx43/ZO-1 binding on microtubule targeting to the membrane was tested in an inducible cardiospecific mouse model, where wild type Cx43 was replaced by Cx43 lacking the PDZ binding domain (Cx43D378stop) [26]. The Cx43D378stop mice died in ventricular fibrillation
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approximately 21 days after induction. The underlying cause was most likely a reduction in INa , IK and a loss of NaV 1.5 from the ID. The decrease in INa was expected if the NaV 1.5 vesicles failed to reach the membrane due a disruption of the linkage between the ID and the microtubule. This hypothesis was directly tested on the D378stop mouse cardiomyocytes using super resolution microscopy [7]. The study showed that deletion of the PDZ binding domain reduced the number of microtubule plus ends (determined by EB1 localization) that made it to the ID. Consistent with the idea that NaV 1.5 is transported along the microtubules, a similar reduction in the fraction of NaV 1.5 clusters at the ID was observed.
Conclusion Studies demonstrate that the ID is a highly complex structure and that its components interact both functionally and structurally. One important aspect is the proper delivery of the components to the ID and the evidence presented in this review places Cx43 at the crux of delivery via microtubules. Cx43 seems crucial for microtubule organization at the ID, thereby ensuring delivery of both sodium and potassium channels.
Funding This work was supported by the Danish National Research Foundation to L.W.S., C.F.J. and M.S.N.
References 1 Eberth, C.J. (1866) Die Elemente der quergestreiften Muskeln. Archiv fur ¨ pathologische Anatomie und Physiologie und fur ¨ klinische Medicin 37, 100–124 2 Sjostrand, F.S. and Andersson, E. (1954) Electron microscopy of the intercalated discs of cardiac muscle tissue. Experientia. 10, 369–370 CrossRef PubMed 3 Kline, C.F. and Mohler, P.J. (2013) Evolving form to fit function: cardiomyocyte intercalated disc and transverse-tubule membranes. Curr. Top. Membr. 72, 121–158 CrossRef PubMed 4 Nielsen, M.S., Axelsen, L.N., Sorgen, P.L., Verma, V., Delmar, M. and Holstein-Rathlou, N.H. (2012) Gap junctions. Compr. Physiol. 2, 1981–2035 CrossRef PubMed 5 Franke, W.W., Borrmann, C.M., Grund, C. and Pieperhoff, S. (2006) The area composita of adhering junctions connecting heart muscle cells of vertebrates. I. Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins. Eur. J. Cell Biol. 85, 69–82 CrossRef PubMed 6 Rhett, J.M., Ongstad, E.L., Jourdan, J. and Gourdie, R.G. (2012) Cx43 associates with Na(v)1.5 in the cardiomyocyte perinexus. J. Membr. Biol. 245, 411–422 CrossRef PubMed 7 Agullo-Pascual, E., Lin, X., Leo-Macias, A., Zhang, M., Liang, F.X., Li, Z., Pfenniger, A., Lubkemeier, I., Keegan, S., Fenyo, D. et al. (2014) Super-resolution imaging reveals that loss of the C-terminus of connexin43 limits microtubule plus-end capture and NaV1.5 localization at the intercalated disc. Cardiovasc. Res. 104, 371–381 CrossRef PubMed
8 Peters, N.S., Coromilas, J., Severs, N.J. and Wit, A.L. (1997) Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia. Circulation 95, 988–996 CrossRef PubMed 9 Marcus, F.I., Fontaine, G.H., Guiraudon, G., Frank, R., Laurenceau, J.L., Malergue, C. and Grosgogeat, Y. (1982) Right ventricular dysplasia: a report of 24 adult cases. Circulation 65, 384–398 CrossRef PubMed 10 Calore, M., Lorenzon, A., De Bortoli, M., Poloni, G. and Rampazzo, A. (2014) Arrhythmogenic cardiomyopathy: a disease of intercalated discs. Cell Tissue Res, DOI 10.1007/s00441-014-2015-5 CrossRef 11 van Hengel, J., Calore, M., Bauce, B., Dazzo, E., Mazzotti, E., De Bortoli, M., Lorenzon, A., Li Mura, I.E., Beffagna, G., Rigato, I. et al. (2013) Mutations in the area composita protein alphaT-catenin are associated with arrhythmogenic right ventricular cardiomyopathy. Eur. Heart J. 34, 201–210 CrossRef PubMed 12 Kaplan, S.R., Gard, J.J., Protonotarios, N., Tsatsopoulou, A., Spiliopoulou, C., Anastasakis, A., Squarcioni, C.P., McKenna, W.J., Thiene, G., Basso, C. et al. (2004) Remodeling of myocyte gap junctions in arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease). Heart Rhythm 1, 3–11 CrossRef PubMed 13 Noorman, M., Hakim, S., Kessler, E., Groeneweg, J.A., Cox, M.G., Asimaki, A., van Rijen, H.V., van Stuijvenberg, L., Chkourko, H., van der Heyden, M.A. et al. (2013) Remodeling of the cardiac sodium channel, connexin43, and plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy. Heart Rhythm 10, 412–419 CrossRef PubMed 14 Cerrone, M., Lin, X., Zhang, M., Agullo-Pascual, E., Pfenniger, A., Chkourko Gusky, H., Novelli, V., Kim, C., Tirasawadichai, T., Judge, D.P. et al. (2014) Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype. Circulation 129, 1092–1103 CrossRef PubMed 15 Giepmans, B.N., Verlaan, I., Hengeveld, T., Janssen, H., Calafat, J., Falk, M.M. and Moolenaar, W.H. (2001) Gap junction protein connexin-43 interacts directly with microtubules. Curr. Biol. 11, 1364–1368 CrossRef PubMed 16 Francis, R., Xu, X., Park, H., Wei, C.J., Chang, S., Chatterjee, B. and Lo, C. (2011) Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics. PLoS One 6, e26379 CrossRef PubMed 17 Shaw, R.M., Fay, A.J., Puthenveedu, M.A., von Zastrow, M., Jan, Y.N. and Jan, L.Y. (2007) Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions. Cell 128, 547–560 CrossRef PubMed 18 Patel, D.M., Dubash, A.D., Kreitzer, G. and Green, K.J. (2014) Disease mutations in desmoplakin inhibit Cx43 membrane targeting mediated by desmoplakin-EB1 interactions. J. Cell Biol. 206, 779–797 CrossRef PubMed 19 Jansen, J.A., Noorman, M., Musa, H., Stein, M., de Jong, S., van der Nagel, R., Hund, T.J., Mohler, P.J., Vos, M.A., van Veen, T.A. et al. (2012) Reduced heterogeneous expression of Cx43 results in decreased Nav1.5 expression and reduced sodium current that accounts for arrhythmia vulnerability in conditional Cx43 knockout mice. Heart Rhythm 9, 600–607 CrossRef PubMed 20 Desplantez, T., McCain, M.L., Beauchamp, P., Rigoli, G., Rothen-Rutishauser, B., Parker, K.K. and Kleber, A.G. (2012) Connexin43 ablation in foetal atrial myocytes decreases electrical coupling, partner connexins, and sodium current. Cardiovasc. Res. 94, 58–65 CrossRef PubMed 21 Petitprez, S., Zmoos, A.F., Ogrodnik, J., Balse, E., Raad, N., El-Haou, S., Albesa, M., Bittihn, P., Luther, S., Lehnart, S.E. et al. (2011) SAP97 and dystrophin macromolecular complexes determine two pools of cardiac sodium channels Nav1.5 in cardiomyocytes. Circ. Res. 108, 294–304 CrossRef PubMed 22 Lowe, J.S., Palygin, O., Bhasin, N., Hund, T.J., Boyden, P.A., Shibata, E., Anderson, M.E. and Mohler, P.J. (2008) Voltage-gated Nav channel targeting in the heart requires an ankyrin-G dependent cellular pathway. J. Cell Biol. 180, 173–186 CrossRef PubMed 23 Makara, M.A., Curran, J., Little, S.C., Musa, H., Polina, I., Smith, S.A., Wright, P.J., Unudurthi, S.D., Snyder, J., Bennett, V. et al. (2014) Ankyrin-G coordinates intercalated disc signaling platform to regulate cardiac excitability in vivo. Circ. Res. 115, 929–938 CrossRef PubMed C The
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24 Casini, S., Tan, H.L., Demirayak, I., Remme, C.A., Amin, A.S., Scicluna, B.P., Chatyan, H., Ruijter, J.M., Bezzina, C.R., van Ginneken, A.C. and Veldkamp, M.W. (2010) Tubulin polymerization modifies cardiac sodium channel expression and gating. Cardiovasc. Res. 85, 691–700 CrossRef PubMed 25 Rhett, J.M., Jourdan, J. and Gourdie, R.G. (2011) Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1. Mol. Biol. Cell 22, 1516–1528 CrossRef PubMed
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26 Lubkemeier, I., Requardt, R.P., Lin, X., Sasse, P., Andrie, R., Schrickel, J.W., Chkourko, H., Bukauskas, F.F., Kim, J.S., Frank, M. et al. (2013) Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels. Basic Res. Cardiol. 108, 348 CrossRef PubMed Received 25 February 2015 doi:10.1042/BST20150059
Biochemical Society Transactions (2015) Volume 43, part 3
Gap junctions – guards of excitability Line Waring Stroemlund*1 , Christa Funch Jensen*, Klaus Qvortrup†, Mario Delmar‡ and Morten Schak Nielsen* *Department of Biomedical Sciences and The Danish National Research Foundation Centre for Cardiac Arrhythmia, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark †Department of Biomedical Sciences and Core Facility for Integrated Microscopy, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark ‡Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, U.S.A.
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although these structures have long been known, it is becoming increasingly clear that their components interact. This review describes the involvement of the ID in electrical disturbances of the heart and focuses on the role of the gap junctional protein connexin 43 (Cx43). Current evidence shows that Cx43 plays a crucial role in organizing microtubules at the intercalated disc and thereby regulating the trafficking of the cardiac sodium channel NaV 1.5 to the membrane.
The intercalated disc The efficient activation and contraction of the heart requires that cardiomyocytes are coupled both electrically and mechanically, which occurs in the intercalated disc (ID). The ID was first observed by light microscopy in 1866 by Eberth [1]. In 1954 Sjostrand and Andersson [2] imaged the ID at high resolution using electron microscopy (EM), demonstrating its complex nature. Figure 1 shows an overview and details of a rat ID imaged by EM. The ID is a highly organized structure (Figures 1 and 2), composed of both anchoring complexes (fascia adherens junctions and desmosomes) and sites of intercellular communication (gap junctions). The mechanical connections in adherens junctions and desmosomes both include proteins of the cadherin family. The cadherin family is a class of proteins that dock with cadherins from neighbouring cells in a calcium dependent manner. On the intracellular side they link to the contractile machinery and intermediate filaments, and thus ensure both efficient transfer of mechanical force from cell to cell and mechanical stability of the cells (for review see Kline and Mohler [3]). The adherens junctions consist of the transmembrane protein N-cadherin. The extracellular domain is responsible for the calcium dependent binding to cadherins in neighbouring cells, whereas the intracellular domain is linked to the actin cytoskeleton through a complex involving α-catenin, βcatenin and vinculin [3]. Desmosomes consist of desmocollins and desmogleins that bind to each other through the extracellular domain. On the intracellular side, they form complexes with desmoplakin,
Key words: Cx43, intercalated disc, microtubule, Nav1.5, trafficking. Abbreviations: ACM, arrhythmogenic cardiomyopathy; AnkG, ankyrin-G; Cx43, connexin 43; EB1, plus-end tracking protein; EM, electron microscopy; ID, intercalated disc; IK , potassium current; INa , sodium current; NaV 1.5, sodium channel; PKP2, plakophilin-2; ZO-1, zonula occludens protein-1. 1 To whom correspondence should be addressed (email [email protected]).
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plakophilin-2 and plakoglobin and anchors to the desmin based intermediate filaments [3]. Gap junctions are composed of intercellular channels that allow passage of small ions and molecules between neighbouring cells. The channels are formed by proteins of the connexin family, where connexin 43 (Cx43) is the dominant isoform in the working myocardium. Six connexins form a connexon and when two connoxons from neighbouring cells dock in the extracellular space, they form an intercellular pore [4]. Although the adherens junctions, desmosomes and gap junctions are discernible as separate structures in EM, mixing of proteins from the adherens junctions and desmosomes has been reported. These areas of mixed-type junctions are referred to as the area composita [5]. Likewise, Cx43 has been located outside the gap junctional structure [6,7].
Intercalated disc diseases Several pathologies are related to mutations in ID components and the functionality of the ID. Some of these are acquired diseases, such as infarction, and associated with disarray of ID components, a disarray believed to contribute to arrhythmogenesis [8]. Acquired diseases are often complex, making it difficult to establish the exact causality, but lessons taught from rare genetic diseases have helped improve our current understanding of ID function. Arrhythmogenic cardiomyopathy (ACM) is formerly referred to as arrhythmogenic right ventricular cardiomyopathy (ARVC). The disease is characterized by fatty or fibrofatty replacement predominantly in the right ventricle which can lead to ventricular arrhythmias, ventricular dysfunction and cause of sudden cardiac death [9]. ACM has previously been considered a desmosomal disease as mutations were primarily found in the components of the desmosome, including plakophilin-2 (PKP2 gene), Biochem. Soc. Trans. (2015) 43, 508–512; doi:10.1042/BST20150059
8th UK Gap Junction Meeting
Figure 1 Electron micrograph of intercalated disc between rat cardiomyocytes (A) Electron micrograph showing an overview image of an intercalated disk (ID) between two rat myocytes, illustrating its stair-like structure, with fascia adherens (FA) running perpendicular to the fibre orientation and gap junctions (GJ) running in parallel often followed by a desmosome (D). (B) Magnified detail image of fascia adherens (FA), desmosome (D) gap junctions (GJ), and perinexus (PN).
Figure 2 Schematic representation of the cardiac intercalated disc components Simplified model of the molecular composition of the cardiac intercalated disc, including components of the desmosome (D), fascia adherens (FA), the cardiac sodium channel NaV 1.5, gap junction (GJ) and the cytoskeleton.
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desmoglein-2 (DSG2 gene), desmocollin-2 (DSC2 gene), desmoplakin (DSP gene) and plakoglobin (the JUP gene) [10]. However, ACM may also be caused by mutations in proteins localized outside the desmosome. Van Hengel et al. [11] screened 76 ACM patients without mutations in desmosomal genes and found two mutations in the CTNNA3 gene encoding αT-catenin which is expressed in the adherens junctions. Since then, ACM causing mutations have been described in additional ID components [10]. ACM can therefore no longer be considered as a purely desmosomal disease but rather a disease of the ID. Furthermore, mutations in components of the mechanical junctions also affect structures involved in the propagation and initiation of the cardiac action potential. Saffitz and co-workers showed that plakoglobin mutations causing Naxos disease were associated with significant gap junction remodelling [12]. In a comprehensive study of 20 patients, Noorman et al. [13] showed that not only Cx43 but also NaV 1.5 were severely affected in a large fraction of ACM patients. These findings indicate that gap junction and sodium channel remodelling is likely a common trait in ACM. More recently, it was shown that mutations in structural ID proteins need not lead to ACM but may result in electrical abnormalities without gross anatomical alterations. Specifically, Cerrone et al. [14] showed that a missense mutation leading to loss of PKP-2 reduced NaV 1.5 expression at the ID and decreased sodium current (INa ), which explained the Brugada syndrome phenotype of patients carrying the mutation. All of these observations point to the fact that alterations in any ID protein are likely to affect the entire ID. This in turn raises the question of the exact mechanisms that govern the localization and function of key proteins such as Cx43 and NaV 1.5. The gap junctional protein Cx43 is known to function, not only in its capacity of forming intercellular pores, but also as hub binding a multitude of partners. Evidence now shows that Cx43 may be a major organizer of transport to the ID.
Cx43 and the microtubular network Giepmans et al. [15] were the first to show that the Cterminal tail of Cx43 interacts directly with tubulin, and that microtubules preferentially terminates in areas of cell to cell contact. This led the authors to suggest that gap junctions act as anchoring points for microtubules. Since then studies have confirmed and elaborated on the Cx43 interactions with the microtubular network. Lo and coworkers showed that cytoskeletal organization and cell migration was dependent on the presence of Cx43 and that the deletion of a tubulin-binding motif recapitulated the phenotype of cells lacking Cx43 [16]. Based on these studies it seems that Cx43 is a key regulator of microtubule organization. However, the exact nature of microtubule binding to the ID still remains uncertain. Some studies suggest that the microtubule is interacting directly with Cx43 via
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microtubule binding domains [15,16], while other studies suggest that that microtubule plus-end tracking protein EB1 interacts (possibly via a secondary interactions) with Cx43 [7], adherens junctions [17] or desmoplakin [18]. Irrespective of the nature of EB1 binding, Cx43 seems to play an important role in the final stabilization of microtubules at the membrane. The interaction between the microtubule and Cx43 potentially affects not only Cx43 trafficking, but also the trafficking of other ion channels. This notion is supported by Jansen et al. showing that knockout of Cx43 reduced the expression of NaV 1.5, reduced INa in mouse ventricular myocytes, and made hearts more prone to ventricular arrhythmias [19]. Desplantez et al. [20] made similar observations on atrial myocytes from mice with genetic ablation of Cx43 showing decreased INa and demonstrating that the interaction between Cx43 and NaV 1.5 is not restricted to ventricular myocytes. NaV 1.5 is the main cardiac voltage-gated Na + channel and is found preferentially at the ID but also to lesser extent in costameric and T-tubular locations. The trafficking basis for this distribution is still unclear but membrane expression of NaV 1.5 depends at least partially on both SAP97 [21] and ankyrin-G (AnkG) [22]. The in vivo importance of AnkG was recently demonstrated by Mohler and co-workers, who showed that knockout of AnkG reduced NaV 1.5 specifically at the ID [23]. These findings do, however, not resolve the pathway by which NaV 1.5 is trafficked to the membrane, but Casini et al. [24] showed that interfering with microtubule polymerization led to reduced surface expression. This leaves Cx43 as an interesting candidate for leading microtubules and thereby NaV 1.5 to the ID. This hypothesis was directly addressed by Agullo-Pascual et al. [7] using HL1 cells expressing either wild type Cx43, Cx43 lacking the microtubule binding domain or lacking Cx43 altogether. The study showed that removing Cx43 entirely or removing the microtubule binding domain both reduced INa to a similar extent. The exact location of Cx43 microtubule interaction is unknown, but since it is unlikely that NaV 1.5 is delivered inside the gap junction plaques, the interaction probably occurs outside the plaque itself. One such location is the perinexus at the transition between the gap- and adherens junction (see Figure 1B) where Cx43 and NaV 1.5 colocalize [6]. More recently it was demonstrated that Cx43 is also located throughout the adherence junctions, where they (due to membrane spacing) are unlikely to form intercellular channels but could serve the function of anchoring microtubules [7]. At least in the perinexus Cx43 interacts with its scaffolding protein zonula occludens protein-1 (ZO-1) through a PDZ binding domain located in the last five amino acids of the C-terminus [25]. The influence of Cx43/ZO-1 binding on microtubule targeting to the membrane was tested in an inducible cardiospecific mouse model, where wild type Cx43 was replaced by Cx43 lacking the PDZ binding domain (Cx43D378stop) [26]. The Cx43D378stop mice died in ventricular fibrillation
8th UK Gap Junction Meeting
approximately 21 days after induction. The underlying cause was most likely a reduction in INa , IK and a loss of NaV 1.5 from the ID. The decrease in INa was expected if the NaV 1.5 vesicles failed to reach the membrane due a disruption of the linkage between the ID and the microtubule. This hypothesis was directly tested on the D378stop mouse cardiomyocytes using super resolution microscopy [7]. The study showed that deletion of the PDZ binding domain reduced the number of microtubule plus ends (determined by EB1 localization) that made it to the ID. Consistent with the idea that NaV 1.5 is transported along the microtubules, a similar reduction in the fraction of NaV 1.5 clusters at the ID was observed.
Conclusion Studies demonstrate that the ID is a highly complex structure and that its components interact both functionally and structurally. One important aspect is the proper delivery of the components to the ID and the evidence presented in this review places Cx43 at the crux of delivery via microtubules. Cx43 seems crucial for microtubule organization at the ID, thereby ensuring delivery of both sodium and potassium channels.
Funding This work was supported by the Danish National Research Foundation to L.W.S., C.F.J. and M.S.N.
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