Heart Failure Reviews (2019) 24:115–132 https://doi.org/10.1007/s10741-018-9743-7

Intercalated discs: cellular adhesion and signaling in health and diseases

Guangze Zhao1,2 & Ye Qiu3 & Huifang M. Zhang 1,2 & Decheng Yang1,2

Published online: 5 October 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Intercalated discs (ICDs) are highly orchestrated structures that connect neighboring cardiomyocytes in the heart. Three major complexes are distinguished in ICD: , (AJ), and (GJ). are major cell adhesion junctions that anchor cell membrane to the network; AJs connect the cytoskeleton of adjacent cells; and gap junctions metabolically and electrically connect the cytoplasm of adjacent cardiomyocytes. All these complexes work as a single unit, the so-called area composita, interdependently rather than individually. Mutation or altered expression of ICD proteins results in various cardiac diseases, such as ARVC (arrhythmogenic right ventricular ), dilated cardiomyopathy, and hypotrophy cardiomyopathy, eventually leading to . In this article, we first review the recent findings on the structural organization of ICD and their functions and then focus on the recent advances in molecular pathogenesis of the ICD-related heart diseases, which include two major areas: i) the ICD gene mutations in cardiac diseases, and ii) the involvement of ICD proteins in signal transduction pathways leading to myocardium remodeling and eventual heart failure. These major ICD-related signaling pathways include Wnt/β-catenin pathway, p38 MAPK cascade, Rho-dependent serum response factor (SRF) signaling, calcineurin/NFATsignaling, Hippo kinase cascade, etc., which are differentially regulated in pathological conditions.

Keywords Intercalated disc . Desmosome . Adherens junction . Gap junction . Signaling . Cardiomyopathy

Introduction cells; AJ and desmosome provide mechanical attachment be- tween two cardiomyocytes by anchoring the actin cytoskele- Normal heart function requires the synchronous mechanical ton and intermediate filaments (IF), respectively, which en- and electrical activity of individual cardiomyocytes to ensure hances the strength and stability of the myocardium. Studies the coordinated excitation and contractile performance of the have shown that these three complexes do not function inde- myocardium. The intercalated disc (ICD), a highly organized pendently but co-operate with each other via cross-talking cell-cell adhesion structure connecting a cardiomyocyte to one between mechanical and electrical junctions at the ICD. another, fulfills this important role [1, 2]. ICD is composed of Thus researchers have renamed the ICD protein interactions three major complexes, gap junction (GJ), desmosome, and into adhering junction, the so called area composita [3]. adherens junction (AJ). GJ connects the cytoplasm of adjacent Due to the important roles of ICD, it is not surprising that cardiomyocytes metabolically and electronically to enable the mutation or defect of ICD proteins causes a range of heart propagation of electrical stimuli throughout the heart muscle diseases, such as arrhythmogenic right ventricular cardiomy- opathy (ARVC), hypertrophy cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) [2, 4, 5]. In addition, recent * Decheng Yang studies provided a large amount of data showing that ICD [email protected] proteins are involved in regulating signal transduction path- ways leading to cardiac remodeling and diseases [6, 7]. In this 1 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada review, we will first discuss the recent progresses of research on structural organization and functions of ICD proteins as 2 Center for Heart Lung Innovation - St. Paul’s Hospital, University of British Columbia, 1081 Burrard Street, Vancouver, BC V6Z 1Y6, well as their mutations in causing heart diseases. Secondly, Canada we will also focus on the activation or suppression of the 3 College of Biology, Hunan Universsity, Changsha, China signaling pathways in various myocardial disorders caused 116 Heart Fail Rev (2019) 24:115–132 by the altered levels of these ICD component proteins. We catenin) and plakophilin (Pkp-2). Pkg is also found in AJ. notice that several reviews have been recently published [8, Pkg contains 12 arm repeats, which share 65% identity with 9]. However, our review is different from them by focusing on the one present in β-catenin, and are flanked by Pro-Lys-Gly- mutational causes of ICD-related inherited heart diseases and rich N- and C-terminal domains [14–16]. Pkg interacts with signal transduction pathways associated with ICD proteins. In desmosomal cadherins via its N-terminal domain, and the arm addition, wherever possible, we mention the virus-induced repeats near its C-terminus. Although it can interact with both ICD destruction and alteration of the cellular signaling. desmosomal and AJ cadherins, it has a higher affinity to Dsg- 2, supporting its main desmosomal localization [17]. In addi- tion, Pkg can also interact via its central arm repeats with ICD structural organization and function , which further binds to -containing IFs. Plakophilin has four splicing products, but Pkp-2 is the most As mentioned above, ICDs, as highly organized units between prominent form in human cardiomyocytes. Pkp-2 binds to cardiomyocytes, maintain structural integrity and synchro- several desmosomal proteins via its N-terminal domain, in- nized function of the heart. The ICD is categorized into three cluding desmocollin, desmoplakin, and as well major junctional complexes (Fig. 1): the desmosome, which as actin and the IF proteins (desmin and keratin) [18]. functions as a cell anchor, the AJ, which provides cell Furthermore, Pkp-2 also interacts with Ankyrin-G, a sodium strength, and the GJ, which couples cells electrically and met- channel-anchoring protein, and with connexin 43 (Cx43) [19], abolically. For cellular communication, AJs and desmosomes a major component protein of GJ. Besides Ankyrin-G, Pkp-2 are tightly connected to the cytoskeleton. Furthermore, several interacts with PKCα, which is necessary for phosphorylation proteins that are not involved in direct cell-cell contact such as and recruitment of Dsp to newly forming desmosomes during ion channels also reside in the ICD. Here, we will only focus heart development or repair of heart injury [11]. on the discussion of the three major complexes: Desmoplakin and myozap Desmoplakin (Dsp) is a large Desmosome multi-domain protein that links the IF desmin and desmo- somes. It is the major plakin protein expressed in the heart, Desmosomes are intercellular adhesions and part of the ICDs. and its structure is characterized by a central α-helical coiled- Desmoglein and desmocollin, the two cadherin-type trans- coil rod domain, which is flanked by globular N- and C-ter- membrane adhesion molecules, build up the desmosomal core mini. Through its α-helical region, Dsp has been suggested to and are linked to the cytoskeleton via adapter proteins, such as form homodimers. Its N-terminus binds to Pkg and Pkp-2, plakoglobin, plakophilin, and desmoplakin. Four desmoglein targeting them to desmosomes [20], while its C-terminal tail isoforms and three desmocollin isoforms have been identified containing three plakin-repeat domains mediates binding to so far, and the expression of them shows a distinct tissue- desmin protein [21, 22]. Thus, like other desmosomal protein, specific distribution pattern [10], with desmoglein-2 (Dsg-2) Dsp-2 plays an important role in desmosome assembly. and desmocollin-2 (Dsc-2) being the main isoforms expressed Myozap (myocardium-enriched zonula occludens-1- in cardiomyocytes [11]. interacting protein) is a recently identified ICD protein, which is highly expressed in the heart. In ICD, it colocalizes with β- Dsg-2 and Dsc-2 Dsg-2 and Dsc-2 are members of the super- catenin, N-cadherin as well as Pkp-2 and directly binds to Dsp family of Ca2+-dependent desmosomal adhesion molecules, and zonula occludens 1 (ZO-1). Knockdown of myozap in which form dimers via heterophilic interactions to make up zebrafish results in cardiomyopathy with severe contractile the core of desmosomal junctions [12]. They are highly ho- dysfunction [23]. Overexpression of myozap in mice sensi- mologous and share ~ 30% identity in the amino acid se- tizes the animals to biomechanical stress and develops quence [13]. The majority of the homology is found within protein-aggregate-associated cardiomyopathy [24]. their extracellular domains, while their non-homologous re- gions are found within the C-termini. Within the desmosome, Adherens junction the intracellular tails of these desmosomal cadherins associate with armadillo (arm) proteins, plakoglobin, and plakophilin, N-cadherin AJ, also called fascia adherens, is the primary while their intercellular portions interact with the counterparts anchor for and connects actin filaments from adja- of the desmosomal cadherins from the adjacent cent cardiomyocytes, which provides structural support for the cardiomyocytes. heart muscle cells. In addition, it transduces signals concerning actin cytoskeleton and senses mechanical forces Plakoglobin and plakophilin-2 Desmosomal cadherins form on the cells [25, 26]. N-cadherin (N-cad), also known as cytoplasmic connections with IFs partially via proteins of ar- cadherin-2, is a member of the classical cadherin superfamily madillo family including plakoglobin (Pkg, also named γ- of transmembrane glycoproteins, including epithelial (E)-, Heart Fail Rev (2019) 24:115–132 117

Fig. 1 ICD structure. Three ICD complexes, desmosome, adherens junction, and gap junction, are located between two cardiomyocytes. Certain component proteins are present in more than one position, suggesting that ICD proteins form an interactional network and function as a single unit placental (P)-, and neural (N)-cadherin. N-cad, the sole This provides tissue specificity during development, allowing cadherin expressed in the heart, is a single-pass transmem- cells to interact only with cells expressing the same cadherin. brane glycoprotein that mediates calcium-dependent cell-cell adhesion [27]. It forms homodimers with N-cad from adjacent β-catenin and other (γ-, α-, and p120-) catenins Catenins can cells in the extracellular space, as an intercellular zipper [2]. be divided into two families: the armadillo domain-containing catenins (β-catenin, γ-catenin/plakoglobin, and p120 catenin) 118 Heart Fail Rev (2019) 24:115–132 that bind directly to the N-cad and the -homology location within ICD and its interaction with the AJ protein, β- domain-containing catenins (αE-catenin and αT-catenin). β- catenin, and the GJ proteins, Cx45, and ZO-1. catenin directly binds to the C-terminal cytoplasmic domain of N-cad. By associating with α-catenin and vinculin, β-catenin Gap junction connects AJs to the actin cytoskeleton [25]. Also, β-catenin plays a central role in cadherin-mediated signal transduction Cx43 GJ is an agglomeration of multiple individual GJ chan- (see later discussion). p120 catenin also plays an essential role nels with associated proteins. It mediates the electrical and in signaling pathways. It binds the cytoplasmic domain of N- metabolic coupling of adjacent cardiomyocytes by allowing cad close to the membrane. There, it regulates adhesion and passive diffusion of various small molecules (< 1 kDa), such cell shape by binding to the guanine nucleotide exchange as metabolites, water, ions, and microRNAs [21, 37]. factor Vav2, which activates the Rho family GTPase Rac1 Therefore, they warrant electrical and metabolic communica- and Cdc42 [25]. p120 catenin also binds to PLEKHA7 and tion between cells. GJs are present in nearly all tissues of the Nezha, two proteins that mediate the binding of the minus body, but, in heart muscle, GJs ensure a proper propagation of ends of microtubules, thereby ensuring the connection of electrical impulse which triggers sequential and coordinated AJs to microtubules [28]. α-catenins are key cytoplasmic mol- contraction of cardiomyocytes [38]. In general, a GJ channel ecules linking N-cad to actin cytoskeleton via binding to β- in ventricular cardiomyocyte is composed of 12 Cx43 mono- catenin, γ-catenin, vinculin, or actinin. Vinculin is ubiquitous- mers, six of which form a semi-channel (called connexon) in ly expressed membrane-associated protein that links cell ma- the plasma membrane from each cell. One connexon docks to trix adhesions, cell-cell contacts and to the actin another connexon of the opposing membrane in the intercel- cytoskeleton. Our group recently demonstrated that lular space to form a complete AJ channel [2]. Human coxsackievirus infection of mouse heart downregulates vincu- connexin super-family is composed of at least 21 members. lin expression via targeting its gene by microRNA-21 and The Cx43 is the predominant form expressed in the heart leads to destruction of ICDs [29]. α-catenin is different sig- while Cx40 and Cx45 are present in lower amounts [39]. In nificantly in both sequence and structure from other catenins. myocardium, connexins are regionally expressed. Cx43 is Among the different α-catenin isoforms, αT-catenin is the found throughout the heart, except for the nodal tissues and most prominent in the mammalian heart and locates to the parts of the conduction system. Cx45 is preferentially ICD [30]. αT-catenin has also been reported to interact with expressed in the sinoatrial and atrioventricular nodes, but co- Pkp-2 in desmosome [31]. In addition to its role in AJ, α- expressed with Cx43 in the bundle of His and the Purkinje catenin participates in interaction with ZO-1, which is also fibers. Conversely, Cx43 is primarily present in ventricles, but complexed with connexin 43 (Cx43) at GJ [32]. also co-expressed with Cx40 in the atria [40].

Xin-α,LIMP2,spectrin,andCARBesides the classical compo- ZO-1 caveolin, tubulins, and Mena Other proteins associated nent proteins of AJ, several new associated proteins, including with GJs are ZO-1, caveolin, tubulins, Mena, etc. They are Xin-α, LIMP2 (lysosomal integral membrane protein 2), considered as direct binding proteins of GJ channel. ZO-1 spectrin and CAR (coxsackievirus and adenovirus receptor), interacts with Cx43 via its PDZ2 domain that directly binds have been identified. Mouse Xin proteins directly associate to the C-terminus of Cx43. This interaction often takes place with β-catenin and actin. It colocalizes with N-cad and β- at the periphery of the GJ plaque [41] and plays a role in catenin, suggesting a role of Xin in cell-cell adhesion and targeting or retaining Cx43 to the ICD as well as in regulating Wnt/β-catenin/N-cadherin-mediated signaling and organizing the size of GJ or internalization of Cx43 [42]. Caveolins are actin filament assembly [33]. Xin-α also contains p120 cholesterol-binding membrane proteins and the main scaffold- catenin-binding domain and is involved in p120-mediated sig- ing components of caveolae in lipid rafts. Although they have naling. Xin-β was reported to function downstream of angio- been found to interact with Cx43 in different cell types includ- tensin II signaling, which modulates hypertrophic response in ing cardiomyocytes [43], the evidence for caveolin localized disease [25]. LIMP2 is considered as an ICD protein based on to ICD is thin and needs to be further studied. Microtubules, as its interactions with N-cad and its ability to modulate interac- the binding partners of CX43, have been verified in different tions between phosphorylated β-catenin and N-cad in cell lines [44]. However, only a handful of studies have shown cardiomyocytes [34]. Spectrin is another N-cad binding pro- the interactions between tubulins and CX43 in the heart. For tein [35]. It forms tetramers to bind actin filaments at their example, one report indicates that EB1, a microtube plus-end distal ends. In the ICD, spectrin probably occurs in the most tracking protein, is required to deliver Cx43 to the ICD [45]. stable tetramer form (αIIβII)2, where αII spectrin is found at Consistent with this, disruption of this interaction during is- the distal Z-discs of myofibrils, closest to the ICD [36]. Lastly, chemia significantly reduces the surface expression of Cx43 at CAR has been identified as a component of ICD based on its ICD [45]. Mammalian-enabled (Mena) protein is an actin- regulatory protein that influences cell motility and adhesion. Heart Fail Rev (2019) 24:115–132 119

It interacts with Cx43 and vinculin via its EVH1 domain. It Desmosomal cadherins, Dsg-2 and Dsc-2, form heterophilic regulates the actin cytoskeleton and , both at cell-cell adhesive interactions. Mutations in Dsg-2 have been ICD as well as at focal adhesions [46]. Through binding Rac- identified as causal to ARVC in a number of studies [57–59, 1, Mena protein can modulate Cx43 remodeling in 80, 81, 86]. Recently, a mutation in the unique region of Dsg-2 cardiomyocytes [47]. that is associated with cardiomyopathy promotes the rapid internalization of Dsg-2 and leads to the loss of dimerization in cardiac cells [87]. Other mutations related to heart disease ICD mutations and diseases in Dsg-2 have been reported to alter the post-translational modification of Dsg-2 itself [88] and phosphorylation of Desmosome mutations and diseases Cx43 [89]. However, recently, Zhang et al. sequenced all 15 exons of Dsg2 from heart tissues of 25 patients who died from It has been reported that comparing to other ICD proteins ARVC and 25 from unexplained death. They identified two desmosomal protein genes are major targets of mutations caus- Dsg-2 mutations in ARVC samples and two mutations in two ing inherited heart diseases, particularly the ARVC. ARVC is a unexplained death samples. They concluded that Dsg-2 mu- primary cardiomyopathy that is most commonly diagnosed tations may not be specific for ARVC and may also relate to after an individual present with arrhythmia symptoms. The the fatal arrhythmic events in patients with a morphologically pathogenesis is that fibrofatty replaces the myocardium pro- normal heart [90]. Mutations that cause either premature trun- gressively and causes ventricular tachycardia and sudden cation or mislocation of Dsc-2 have been identified in autoso- death in young people. It primarily affects the right ventricle, mal dominant ARVC [57, 60, 62, 78, 80, 91]. Especially, a and it may also involve the left ventricle [48, 49]. Genetic truncation mutant of Dsc-2 has also been associated with re- studies found that ~ 50% of ARVC patients carry a mutation cessive ARVC with mild palmoplantar keratoderma and in one of the five desmosomal genes (not including myozap) wooly hair [79]. Based on currently available data, Dsc-2 discussed above [50]. As myozap is a recently identified ICD has a lower mutation rate than Dsg-2. protein present in desmosome, there is a lack of mutation data Pkg (γ-catenin) was the first desmosomal protein to be associated with inherited heart diseases thus far. These muta- identified to associate with a cardiocutaneous subtype of tions are largely autosomal dominant inheritance. They not ARVC. A 2-bp deletion (2157-2158del GT) in Pkg causes a only affect the number, structural integrity and proper locali- C-terminal truncation and was identified in patients with the zation of desmosomes but also of gap junctions, resulting in recessively inherited cardiocutaneous form of ARVC, the so- impaired intercellular conductance and thus the development called Naxos disease (known as diffuse non-epidermolytic of arrhythmias. These identified mutations within the five ma- palmoplantar keratoderma with wooly hair and cardiomyopa- jor desmosomal genes have been extensively reviewed by thy). The truncated mutation of Pkg in Naxos disease is several groups [16, 49, 51–53]. Particularly, a very recent thought to disrupt Pkg’s capability of binding to other desmo- study has analyzed the frequency of genetic variants associat- somal proteins within ICD and therefore alters desmosome ed with ARVC in the genome aggregation database [54]. For structure. Meanwhile, Cx43 protein is also lost from ICD, an updated list of mutations please refer to http://www. which may lead to abnormal electrical coupling of arvcdatabase.info/ [55]or,http://www.ncbi.nlm.nih.gov/ cardiomyocytes and an increase in cardiac arrhythmia associ- omim and Table 1. The following will briefly discuss some ated with this disease [92]. In addition, other mutations were common mutations related to ARVC and other diseases. discovered, which cause ARVC by altering turnover kinetics Among these mutations associated with ARVC develop- ofPkgormissensemutation[59, 93]. ment, ~ 50–70% is present in the gene encoding Pkp-2 de- Dsp is another target of mutation for heart disorders. pending on different cohorts analyzed in the studies [48, 59, Mutations in this gene have been identified as the cause of 69, 75–78]. These account for ~ 20% of diagnosed ARVC Naxos disease, known as Carvajal syndrome [51, 65, 66, 94, cases, while mutations in genes encoding Dsc-2 [56, 59, 79, 95]. Genome-wide linkage analysis of a large Italian family 80] and Dsg-2 [58, 61, 62, 80, 81]accountfor~10–15% of showed that the Dsp mutation in its Pkg-binding site (S299R) cases each [82, 83]. Some other studies reported the even causes an autosomal dominant form of ARVC. Many other higher rate of mutations for Dsg-2 (5–25%) [48, 63, 64, 83]. homozygous and mostly heterozygous mutations in Dsp have ARVC-related missense mutation in Pkp-2 can cause instabil- been detected in patients showing ARVC, and they are rarely ity of the protein via calpain-mediated proteolysis [84]. combined with cutaneous abnormalities [48, 49, 67, 68, 96]. Moreover, truncating Pkp-2 mutations is associated with de- To date, a large body of data has suggested that desmosom- creased Pkp-2 protein expression [57, 70, 71, 85]. Like other al protein mutations are also major causes of DCM and HCM. forms of ARVC, heterozygous mutations in Pkp-2 lead to This notion is supported by a recent multiple center and mul- abnormal GJ Cx43 localization and expression at the ICD tiple national studies through next-generation sequencing of and thus to the weakening of electrical communication. 84 genes from 639 patients. They found that Pkp-2, - 120 Heart Fail Rev (2019) 24:115–132

Table 1 ICD gene mutations and heart diseases*

ICD gene Protein Mutation Disease References

Desmosome DSC2 Desmocollin Truncation, misallocation biventricular ACM [56] Rearrangements, deletion, missense ACM [57] Missense ARVC/D [58] Missense ARVC [59] Missense [60] DSG2 Desmoglein-2 Missense ARVC, SCD [61] Missense ARVC [62] Missense biventricular ACM [63] Missense Recessive ARVC [64] Missense, deletion ARVC [59] Missense, abnormal splice ARVC/D [58] Missense, deletion ACM [57] DSP Desmoplakin Missense Carvajal syndrome [65] Missense Carvajal syndrome [66] Truncation, deletion SCD [67] Duplication, truncation SCD, ARVC [68] Missense ARVC/D [58] Missense, nonsense ARVC [59] PKP-2 Plakophillin-2 Deletion, truncation ARVC [69] Missense, nonsense ARVC [59] Missense, abnormal splice ARVC/D [58] Deletion ACM [57] Abnormal splice ARVC/D [70] Missense, termination ARVC/D [71] JUP Plakoglobin (γ-catenin) Missense ARVC [59] Adherens junction CDH2 N-cadherin Missense ARVC [72] Missense ARVC [73] CTNNA3 αT-catenin Missense ACM [56] Gap junction GJA1 Connexin 43 Missense SCD [74]

*This table includes ICD gene mutations published during the past about 3 years. The earlier data can be found in the following two websites: http:// www.arvcdatabase.info and http://www.ncbi.nlm.nih.gov/omim ARVC/D arrhythmogenic right ventricular cardiomyopathy/dysplasia, ACM arrhythmogenic cardiomyopathy, and SCD sudden cardiac death binding protein C-3 and Dsp have the highest number of [72, 73]. In addition, another missense mutation in αT-catenin known mutations and that Pkp-2, Dsp, Dsc-2 and Dsg-2 are was also found in patients with arrhythmogenic cardiomyop- the 4 of 8 most commonly mutated genes related to DCM and athy (ACM) [56](Table1). Unlike the ARVC phenotype HCM [97]. caused by mutations of desmosomal protein genes, certain genetic modifications of N-cad, α-catenin, Xin-α or β- catenin in animal models lead to DCM without myocyte loss AJ mutations and diseases and inflammation [2]. Overexpression of N-cad in transgenic mice causes DCM phenotype, cardiac calcification and intra- Comparing with desmosomal proteins, AJ proteins have not cardiac thrombus [98]. These mice showed altered ICD struc- been extensively analyzed for mutations related to cardiac tures with increased expression of α-, β-catenin, downregu- diseases. Only very recently, two mutations (c686A > C, lated Cx43, and redistributed vinculin. On the other hand, p.Gln229Pro; c.1219G > A, p.Asp407Asn) have been discov- conditional heart-specific N-cad knockout (KO) causes the ered in N-cad of ARVC patients by whole genome sequencing Heart Fail Rev (2019) 24:115–132 121 absence of identifiable AJs and desmosomes in ICDs, leading Cx43 is also reported to be associated with HCM. Several to modest DCM. These mice also displayed a significant re- studies have described the changes in the number, size and duction of catenins (α-, β-, γ-, and p120-) and GJ proteins distribution of myocardial GJs in hypertrophic heart. In gen- (Cx40 and Cx43) expression, resulting in slow conduction, eral, Cx43 expression appears to be upregulated during the spontaneous ventricular arrhythmias and sudden death initial compensatory phase of HCM, but downregulated and [98–100]. Other studies using genetically modified mice dem- heterogeneously distributed throughout the ventricles in the onstrated that ablation of α-catenin, β-catenin, vinculin genes, decompensated phase of HCM [110, 111]. Recently, the or perturbation of their expression causes ICD abnormalities downregulation of Cx43 expression in cardiomyocytes has and early DCM or death [101, 102]. Interestingly, mutations been linked to the overexpression of several microRNAs of vinculin have been found not only in DCM patients but also (miR-1, −206 and −130a), which directly target the 3′UTR in obstructive HCM patients [103]. In addition, of Cx43 mRNA to suppress its translation [112, 113]. cardiomyocyte-specific CAR KO in mice causes an atrioven- Mutations in other GJ associated proteins have not been well tricular block, which is associated with loss of Cx45, β-caten- studied; however, a few studies on genetic modifications have in, and ZO-1 localization at the ICD, as well as ultrastructural been reported, for example, KO of ZO-1 and caveolin in mice abnormalities leading to DCM [104]. This finding supports causes embryonic lethality and early DCM, respectively [114]. the notion that CAR is implicated in human viral myocarditis and its late stage, DCM [105]. ICD protein disruption and heart dysfunction

In addition to the inherited heart diseases caused by ICD gene GJ mutations and diseases mutations, other heart dysfunctions can be caused by the dam- age of ICD integrity. The causes of this damage can be various Connexins are abundant in GJ, particularly Cx43 in cellular stresses or infections, which lead to the reduction of cardiomyocytes. Although the genetic connection between certain ICD protein levels and in turn the damage of the ICD GJA1 gene (coding for Cx43) mutations and ARVC has not architecture. For example, rat neonatal cardiomyocytes treated been confirmed, a recent study by genomic sequencing of with ionomycin can cause activation of calpains, cleavage of DNA from sudden unexpected nocturnal death syndrome pa- N-cad and decreased expression of β-catenin and Cx43 [115]. tients found that two mutations (c.169C > T; SNP c.624C > Further study using MI/calpastatin (inhibitory gene for T(rs530633057)) in GJA1 gene is potentially associated with calpains) KO mouse model found that calpains are activated arrhythmogenic cardiac disorders [74](Table1). Other genetic during the chronic phase and that profound activation of linkage analyses have implicated connexins in at least 14 hu- calpains exacerbates LV remodeling after MI in these mice man diseases―many of them can be recapitulated in mutant [115]. As for viral infection, our group recently demonstrated connexin mouse models [106]. Cx43, as the major component that infection of mouse heart by coxsackievirus, a major path- of GJ, has been reported to be associated with multiple dis- ogen of human myocarditis, causes desmin protein degrada- eases. Mutations related to the development of tion via the ubiquitin-proteasome-mediated pathway. This oculodentodigital dysplasia (OOOD) that is frequently accom- pathway is activated by microRNA-21 mediated suppression panied by hair and skin defects have been identified in Cx43 of a deubiquitinase, YOD1 [29]. We further showed that gene, too [107]. Some of these mutations are linked to the coxsackievirus infection directly downregulates vinculin pro- development of cardiac disturbances because the Cx43 ex- tein expression in mouse heart by targeting the 3′ untranslated pression levels and the number of GJs are moderately de- region of vinculin mRNA [29]. This disruptions/reduction of creased in myocardium [108]; however, the cardiac conduc- ICD proteins results in disorganization of desmosome and tion is not affected. This means that sole mutation in Cx43 AJs, leading to severe myocarditis, which is evidenced by cannot be the primary determinant of conduction defects un- the increased cardiomyocyte necrosis and inflammatory derlying the arrhythmogenesis. The causal relation between infiltrations. abnormal expression of Cx43 and the heart disease develop- ment is also reflected in HIV-infected patients. HIV infection increases Cx43 expression in the heart. The upregulated Cx43 ICD proteins in signal transduction pathways localizes along the lateral membrane of the cardiomyocyte and in the ICD. Areas of HIV-positive tissue with anomalous The ICD proteins, in addition to playing a critical role in Cx43 expression and localization also show calcium overload, maintaining the integrity and proper function of the heart, also sarcofilamental atrophy, and accumulation of collagen [109]. play an essential role in regulating molecular signal transduc- This suggests that virus-induced upregulation of Cx43 causes tion pathways. Comparing to the investigations of ICD struc- ICD damage that likely contributes to the high rates of cardio- ture and functions, the signal transduction pathways associat- vascular disease in HIV-infected individuals. ed with ICD proteins in health or diseases were less studied. 122 Heart Fail Rev (2019) 24:115–132

However, recently increasing number of reports have en- of EGFR-mediated signaling, decreasing cancer cell migra- hanced our knowledge on the signaling pathways leading to tion, proliferation, and tumor development [131]. Other stud- the disease phenotypes, such as HCM, DCM and ARVC [23, ies showed that knockdown of Pkp-2 in keratinocytes acti- 25, 116, 117]. These studies have provided novel insights into vates RhoA and PKC-α signaling pathway [130], raising the the understanding of the molecular pathogenesis of heart dis- possibility that these similar signaling deficits may contribute eases caused by various cellular stresses [9]. to ARVC pathogenesis. Other signaling pathways related to Dsg-2, Dsc-2, and Dsp were studied expensively in non- Cellular signaling in desmosome cardiomyocytes, which have been summarized in several re- views [7, 51]. Since these data are beyond the focus of this Desmosome proteins, like other ICD components, recently review, we only list the related references in Table 2 without emerged as cell signaling regulators. Loss of expression or further discussion here. interference with the function of desmosome components re- Myozap is a relatively new addition to the ICD protein list sults in diseases of heart and skin. Pkg is probably the most [23]. In ICD, it can directly interact with desmoplakin and ZO- studied signaling protein in desmosome [118]. A recent study 1. More importantly, it can activate the serum response factor reported that Pkg is involved in sympathetic signaling. The (SRF) signaling in a Rho-dependent fashion to link the ICD to sympathetic nervous system via its secondary messenger cardiac gene regulation; however, its another binding partner, cAMP modulates heart function by inducing positive myosin phosphatase-RhoA interacting protein (MRIP), in- adhesiotropic effect [119]. Recent data demonstrated that the hibits this pathway [23]. Further studies by heart-specific β-adrenergic receptor is also located at the ICD and showed overexpression of myozap in mice showed that mice are sen- that desmosomal adhesion is crucial for sufficient adaptation sitized to biomechanical stress and develop a protein- of cardiac myocytes to β-adrenergic signaling [120]. Schinner aggregate-associated cardiomyopathy [24]. However, in et al. found that sympathetic stimulation increases intercellular myozap KO mice, although the structural and functional in- cohesion of cardiac myocytes [121]. Phosphorylation of Pkg tegrity of the heart is not disrupted, the mice display severe at serine 665 by protein kinase A is associated with an in- cellular and cardiac hypertrophy, cardiac remodeling, in- creased force of Dsg-2-mediated binding and recruitment of creased fibrosis, and alterations in hypertrophic signaling cas- Dsg-2, Dsp, and Pkg to the ICD. This effect seems to be cades. Specifically, myozap KO leads to inhibition of MAPK/ important for the positive inotropic effect of sympathetic stim- SRF signaling and activation of β-catenin/GSK-3β and ulation because Pkg deficiency, as well as overexpression of calcineurin/NFAT signaling during pressure overload. This the phosphor-deficient Pkg-mutant S665A, abrogates both indicates that myozap is required for proper adaptation to bio- cAMP-mediated junctional remodeling and increase of cohe- mechanical stress [117]. sion [121]. In addition to Pkg, other desmosome proteins, such as Dsg- Cellular signaling in AJs 2, Dsc-2, Pkp-2, and Dsp, have also been implicated in ARVC and related signaling pathways [9, 122–125](Table2). For As mentioned earlier, N-cad is the sole cadherin that expressed example, suppression of Dsp expression in cardiomyocytes in heart muscle [27]. Although the N-cad-regulated signal leads to the nuclear translocation of Pkg and reduction of pathway has not been well characterized in human heart, it Wnt/β-catenin signaling via inhibition of TCF/LEF1 tran- has been investigated in a murine model and human brain scription factors [133, 135, 136] (Fig. 2). Plakophilins are tissues with Alzheimer disease. The authors demonstrated that another family of desmosome armadillo proteins containing functional disruption of N-cad-mediated cell contact activates three members; Pkp-2, the only Pkp member expressed in p38 MAPK in murine primary neurons, followed by neuronal cardiomyocytes, can indirectly regulate transcription by stim- death. Further, treatment with Aβ42 peptide to decrease the ulating β-catenin/TCF-mediated transcriptional activity, im- expression level of N-cad causes increased phosphorylation of plying a role in Wnt signaling [129, 132, 134]. Loss of Pkp- both p38 MAPK and Tau in murine primary neurons. 2 induces embryonic lethality in a KO mouse model due to Moreover, they showed that phosphorylated levels of p38 heart failure [147]. Like β-catenin described in a later section, MAPK are negatively correlated with that of N-cad in human Pkp proteins have diverse non-desmosomal functions, such as brains. Further, proteomic analyses identified JNK-associated in regulating actin organization, protein synthesis, growth leucine zipper protein (JLP) as a novel interacting protein of control, and disease [6].TakingPkp-2asanexample,itmay N-cad, thus demonstrated a new signaling pathway from N- be the most commonly mutated target in ARVC [148]. This cad to p38 MAPK through the association with JLP [116]. notion can be supported by a study demonstrating the interac- AJ also contains several catenins (α-, β-, γ-, p120-), which tions of Pkp-2 with Cx43 and Ankyrin-G, a cytoskeletal adap- are all related to signaling pathways (Fig. 2). α-catenin has tor protein that regulates the voltage-gated sodium channel been studied extensively in cancer. It is a tumor suppressor complex in the heart [19]. Pkp-2 is also a positive regulator and inhibits several signal molecules: i) inhibits Wnt/β- Heart Fail Rev (2019) 24:115–132 123

Table 2 ICD protein-related sig- naling pathways ICD protein Signaling pathway Cellular effect References

Desmoglein-2 EGFR and ERK1/2 Cell proliferation [122] Cx43 phosphorylation ARVC [89] Desmocollin-2 Wnt/β-catenin Development [123] Akt/ β-catenin Cell proliferation/growth [124] miR-25/β-catenin Transcriptional regulation [125] PKC, PKA, MAPK, and PI3K Differentiation and development [9] Plakoglobin TGFβ JUP-related ARVC [126] Sympathetic signaling Adhesion [120] cAMP, PKA Adhesion [119, 121] PI3K, p38 MAPK Adhesion [127] β-catenin/Hippo/YAP Adiopogenesis [128] Plakophillin-2 Wnt/β-catenin ARVC [129] RhoA /PKC-α Adhesion and migration [130] EGFR activation Proliferation/migration [131] Rho, MAPK Migration/proliferation [132] Cx43/Ankyrin-G Sodium channel function [19] Desmoplakin Wnt/β-catenin ARVC [133, 134] RhoA and PKC-α Skin disease, ARVC [130] EB1/microtubules GJ localization/function [135] γ-catenin/β-catenin, Proliferation, invasion [136] SERCA2/PKCα N-cadherin p38 MAPK/JNK Neuron death [116] α-catenin β-catenin, NFκB, YAP Myocyte proliferation [137] β-catenin Wnt Cardiac remodeling [138] p120-catenin Wnt Cadherin stability, cancer [139] Rac1, Cdc42 Adhesion, cell shape [25] Myozap Rho/SRF Heart hypertrophy, adaptation to cellular [23] stress MAPK/SRF [24] β-catenin/GSK-3β [117] Connexin 43 Akt, MAPK, PKC, Scr GJ assembly/disassembly [140–142] ZO-1 Akt/Cx43 phosphorylation Regulate GJ size [143] CAR Fyn and Ab1 kinase Adhesion [144] Akt/ MAPK/JNK Adhesion [145, 146] Xin-α Wnt/β-catenin/N-cadherin Adhesion, actin assembly [33]

The ICD proteins listed in the table are isoforms expressed in the heart. Certain data are reported so far only from non-cardiomyocytes catenin pathway by preventing β-catenin nuclear transloca- required for β-catenin translocation to the nucleus; and iv) tion or formation of β-catenin-TCF-DNA complex and by the release of the inhibitory action caused by Kaiso transcrip- promoting β-catenin degradation; ii) regulates the Hippo- tional repressor [139]. β-catenin is a multiple functional adap- Yap pathway by blocking YAP dephosphorylation and nucle- tor and plays an important role in development, tissue regen- ar localization; and iii) suppresses the NFκB activity by eration, and disease pathogenesis. Pkg (γ-catenin) is a close inhibiting IkB ubiquitination and degradation [137, 149]. homolog of β-catenin and also locates in AJs. They share 69% However, p120 catenin has a positive effect on the Wnt signal. amino acid identity and many common interacting protein It is involved in four different steps: i) the very early activation partners, such as cadherins, LEF/TCF transcription factors, of CK1ε, essential for Dvl-2 binding to the Wnt receptor com- and the APC/axin degradation machinery. Both β-catenin plex; ii) the internalization of GSK3 and axin into and Pkg are capable of participating in cell signaling in addi- multivesicular bodies, necessary for a complete stabilization tion to their role in cell-cell adhesion. In this context, β-caten- of β-catenin; iii) the activation of Rac1 small GTPase, in, as a transcriptional activator, has a well-documented role as 124 Heart Fail Rev (2019) 24:115–132

Wnt Fzd Lrp

MRIP On-State Axin Desmocollin-2 Desmoplakin

Plakophilin-2 Myozap GSK-3β Dvl Desmoglein-2 CK1 Pkg RhoA CK1

Axin GSK-3β RhoA and PKC-α MAPK/SRF Off-State signaling pathway Signaling Pathway p120 β-cat β-catenin APC N-cadherin β-cat N-cadherin Pkg β-cat Pkg β-cat Rac1-GDP β-cat β-catenin accumulation Rac1-GTP p120 β-catenin Connexin 43 Connexin 43 P ub ub P ub YAP YAP CK-1, PKC, PKA

α-cat Pkg Proteasome

P β-cat Connexin 43 TCF/LEF 1 YAP-TEAD IκB ub Degradation ub ub RELA-NF-κB

Fig. 2 ICD protein-related Wnt signaling and other pathways. The pathways, while the black hammerhead lines indicate inhibitory hollow arrows show the flow of signaling pathways. The red arrows regulation. The black dashed arrows indicate protein translocation indicate stimulatory regulation on the corresponding signaling an important component of the Wnt signaling pathway [150, develop cardiac hypertrophy [154]. Thus, β-catenin- 151]. When the Wnt signal is inactive, the non-junctional β- mediated signaling plays a critical role in cardiac remodeling catenin is rapidly degraded via ubiquitin-proteasome system. in response to stress or injury. The targeting of β-catenin to proteasomes is achieved through Pkg can interact with the same partner proteins and go phosphorylation of β-catenin by a multiple complex through the ubiquitin proteasome-mediated protein degrada- consisting of glycogen synthase kinase-3β (GSK-3β), scaf- tion pathway as β-catenin does, but Pkg also binds to desmo- folding proteins adenomatous polyposis coli (APC), and axin. somal cadherins, Dsc-2 and Dsg-2, in desmosomes, mediating This complex associates with the ubiquitin-proteasome sys- their interactions via Dsp and Pkp-2, with IFs [155]. The abil- tem via the ubiquitin ligase β-TrCP [152]. Phosphorylation of ity of Pkg to transactivate target genes in the nucleus is still β-catenin by GSK-3β targets it for ubiquitination and degra- controversial because it is unable to compensate for defects in dation by 26S proteasome. During the activation of Wnt signal Wnt signaling resulting from lack of β-catenin [156]. via binding to its receptor Frizzled, Wnt ligand activates the However, a number of recent data suggested that Pkg plays scaffolding protein disheveled, which disassembles the multi- opposite (inhibitory) roles in the context of the Wnt pathway ple complexes and inhibits β-catenin turnover by suppression [157–159]. This notion is probably due to the following facts: of GSK-3β activity [153]. This process results in the accumu- i) Pkg may compete with β-catenin signaling by inhibiting lation of β-catenin in the nucleus and its interaction with LEF/ TCF/β-catenin-DNA interactions and Wnt target genes ex- TCF family of transcription factors, which leads to activation pression, ii) Pkg may interact with various cellular partners of target gene expression and cell growth. The terminally dif- involved in signaling and alter their levels, location, and/or ferentiated cardiomyocytes do not require canonical Wnt/ β- functions, and iii) Pkg may interact with transcription factors catenin-mediated signal at least under normal conditions. and regulate gene expression independent of β-catenin [159]. Upon pathological stress, the heart reactivates the Wnt/β- To address these issues proposed above, a number of stud- catenin signaling and develops cardiohypertrophy [138]. ies have been conducted. One of the studies using a However, in heart-specific β-catenin KO mice, the Wnt target cardiomyocyte-restricted Pkg (encoded by JUP gene) mutant gene expression is reduced, and the mice are unable to mouse model demonstrated that ventricular arrhythmias in Heart Fail Rev (2019) 24:115–132 125 this model are associated with progressive cardiomyopathy protein kinase C (PKC) is increased [141, 167]. Moreover, and compensatory fibrosis in JUP-related ARVC and that rapid dephosphorylation of S365 by protein kinase A (PKA) massive cell death plays a crucial role in the initiation of occurs in response to ischemia [168], suggesting that phos- ARVC [126]. Interestingly, it was found that despite the in- phorylation at S365 plays a Bgatekeeper^ role by preventing crease of β-catenin at AJs in JUP mutant cardiomyocytes, the downregulation of gap junctional communication through Wnt/β-catenin signaling is not altered. Further, transforming subsequent Cx43 phosphorylation at S368 by PKC [169]. growth factor-β (TGFβ)-mediated signaling was found sig- However, in rat [166] and rabbit [170], the Cx43 phosphory- nificantly elevated in JUP mutant cardiomyocytes at the early lation at S368 is at baseline in normal condition but signifi- stage of cardiomyopathy. This suggests that TGFβ but not cantly decreases upon ischemia, indicating that PKC interac- Wnt/β-catenin signaling is the pathogenic pathway for JUP- tion with Cx43 shows a species-dependent manner. related ARVC [126]. The effects of Cx43 phosphorylation on GJ assembly/ ARVC is characterized by fibrofatty deposits and nuclear disassembly in non-cardiomyocytes have also been investigated. Pkg has been shown to be necessary for differentiation of There are at least four kinases, Akt, MAPK, PKC, and Scr, cardiac progenitor cells into adipocytes in ARVC [158]. In potentially playing a role in regulation of GJ turnover [140, this disease condition, it has been reported that Pkg can form 142]. Under conditions of injury or growth factor stimulation, a complex with β-catenin and Yes-associated protein (YAP) to Cx43 is phosphorylated by Akt, MAPK, PKC, and Scr, which regulate adipogenesis via the Hippo/YAP signaling pathway induces acute GJ turnover. Salon et al. proposed a model to [128]. This ancient pathway has been linked recently to the describe the sequential phosphorylation of Cx43 by different control of cardiomyocyte proliferation and heart size [160, kinases in response to injury [140]. In either growth factor stim- 161]. The core of the pathway consists of the Hippo kinase ulation or wound, Akt is always the first kinase to phosphorylate cascade, comprising mammalian MST1/2 and LATS1/2. Cx43 at S373 and causes the increase of the GJ size, facilitating MST1/2 phosphorylates and activates LATS1/2, which in turn rapid clearance of Cx43 protein from the plasma membrane. In phosphorylates YAP or WWTR1 (also known as TAZ), tran- this process, ZO-1, a Cx43 binding protein, plays a critical role scriptional coactivators that mediate nuclear responses to by serving as a molecular switch to rapidly increase gap- Hippo signaling. Phosphorylation inhibits YAP and TAZ tran- junctional communication, potentially leading to initiation of ac- scriptional activity by promoting their nuclear export and cy- tivation or ischemic injury response in the heart [143]. Following toplasmic sequestration. Consistent with their role in orches- Akt, MAPK is activated after the accumulation of Cx43 protein trating cell crowd control, the activities of Hippo kinases and in GJ and then phosphorylates Cx43 at S279 and S282, which YAP/TAZ are tightly regulated by cell-cell interactions [128, leads to GJ channel closure. Similarly, activation of PKC leads to 162]. In human keratinocytes, KO of Pkg causes activation of inhibition of new GJ assembly by Cx43 phosphorylation (S368) p38 MAPK-dependent loss of cell adhesion and keratin net- and closure of the GJ channel. In keratinocytes, the next kinase work collapse, which can be rescued by extranuclear Pkg for Cx43 phosphorylation is Scr. Activation of Scr results in expression [127]. phosphorylation of Cx43 at Y247 and initiation of GJ internali- zation. Inhibition of Scr activity with kinase inhibitor PP2 blocks Cellular signaling in GJ growth factor-induced GJ turnover [171]. Scr can directly interact with ZO-1 and compete for binding to the C-terminal region of The essential role of Cx43 in health and disease conditions is Cx43 [172]. Clearly, Scr plays a role in GJ turnover. However, largely associated with its phosphorylation status, which is whether Scr phosphorylation of Cx43 plays a direct role in this controlled by different kinases and phosphatases [140, 163] regard is still unclear. These sequential phosphorylation events (Table 2). Cx43 protein has four transmembrane domains with during wounding are consistent with that during growth factor N- and C-termini on the cytoplasmic side. To date, at least 19/ treatment. Thus, it is obvious that wound-induced GJ disassem- 26 serines and 2/6 tyrosines in the C-terminal region have bly is driven by a kinase program. This program regulates the been identified as phosphorylation sites in cells or tissues accumulation of Cx43 into GJs in preparation for disassembly [140]. In addition, the kinases responsible for the phosphory- and Bmarking^ of specific plaque domains for internalization via lation of Cx43 have also been identified in different cell types, Cx43 phosphorylation at specific residues by these kinases de- such as cardiomyocyte, keratinocyte, osteocyte [86, 142, 164, scribed above [140]. This model supports the notion that a re- 165]. The phosphorylation at different sites by different ki- duction of Cx43 expression and GJ communication can be ben- nases can lead to assembly or disassembly of gap junctions, eficial to wound healing. However, whether these findings on the which depends mainly on the external conditions. In normal kinase program obtained from non-cardiomyocytes are suitable mouse heart, Cx43 is heavily phosphorylated at S325/328/330 for cardiomyocytes needs to be studied. by casein kinase-1 (CK-1), resulting in promoted gap junction In addition to the components of the three major complexes of assembly [102, 164]. But upon ischemia, these CK-1 sits are ICD, CAR protein, the shared receptor by two cardiotropic viruses, dephosphorylated [166] while phosphorylation at S368 by is recently found to be a novel component of GJ based on its 126 Heart Fail Rev (2019) 24:115–132 localization and its interactions with Cx45, ZO-1 as well as β- Dsg-2, which typically locate in desmosome, interact with β-or catenin [104]. CAR is a transmembrane protein (~ 40 kDa) that α-catenin and p120 catenin, respectively, present in AJs [31, functions not only as a docking platform for viral attachment and 134]. Similarly, molecular linkages between desmosomes and entry into host cells but also as an intercellular adhesion molecule GJs have also been identified, for example, Pkp2 and Cx43 to maintain the integrity of ICD structure [104]. Recently, a num- coexist in the same macromolecular complex found within the ber of studies have found that CAR also plays an important role in boundaries of the Cx43 plaque [181] and the relationship be- signal transduction by interactions with different intracellular and tween these two proteins seems to extend to the functional level extracellular proteins [144, 173, 174]. For example, CAR overex- [182]. An additional example is the direct interaction of Dsc-2 pression in human epithelial MCF7 cells increases the activity of with Cx43 [183]. Interactions in the ICD have also been ob- p44/42 MAPK [145]. This upregulation is mediated through the served between mechanical junction proteins and several intracellular domain of CAR, since C-terminal truncated CAR nonjunctional molecules. Such data include the binding and failed to enhance the activation of p44/42. This demonstrates that functional interactions of the voltage-gated sodium channel CAR can regulate signaling pathways and modulate adhesion (NaV1.5) and ankyrin-G (AnkG), a scaffolding protein for the properties leading to increased cell adhesion and paracellular im- sodium channel, with the mechanical junctional proteins, N- permeability. In addition, CAR overexpression in cardiomyocytes cadherin, Pkp2, and Cx43 [184]. Another scaffolding protein can cause an increase in phosphorylation of GSK-3β and Akt ZO-1 present in GJ also mediates the interaction of N-cadherin activation that will lead to cardiomyopathy [146]. and Cx43. ZO-1 can also connect AJs to GJs by interacting with During viral infection, CAR’s role in signal transduction α-catenin via its N-terminal fragment, and with actin via its C- becomes more obvious. In human lung epithelial cells, human terminus [25]. Vinculin anchors actin filaments to the AJ via β- adenovirus-C5 fiber knob (FKAd5) interacts with CAR and catenin. Its head domain also interacts with the PDZ domain of activates p44/p42 MAPK and JNK, as well as the transcription ZO-1, thereby stabilizing GJs, either directly or via α-catenin. factor NF-κB[173]. Activation of p44/p42 MAPK is detected The notion that Cx43, ZO-1, and vinculin form a complex im- by phosphorylation of a peptide substrate upon FKAd5 bind- plies that vinculin is an important anchoring point between AJs ing, FKAd5 also leads to the phosphorylation of JNK1, a and GJs [185]. Taken together, these data indicate that ICD forms MAPK of the JNK/SAPK family within the same time frame a molecular interaction network in which the junctional and non- than p44/p42 MAPK and is abolished if FKAd5 is preincubat- junctional proteins function via cross-talk. This supports the idea ed with recombinant CAR D1. Finally, FKAd5/CAR interac- of ICD being a single functional unit to control the cardiac ex- tion triggers the translocation of the p65 subunit of NF-κB, citability, electric coupling, and intercellular adhesion [186]. which leads to the activation of genes mainly associated with To date, about 200 known proteins are associated with ICD. immune responses. During coxsackievirus type B (CVB) in- This number is rapidly increasing with the application of new fection of the cardiomyocytes, CAR also acts as a primary technologies of proteomic and genomic analyses in the gene receptor for viral entry. CVB3 infects the polarized cells profiling [187, 188]. Among these proteins, 40% of which may through the use of CAR and DAF (decay accelerating factor, have mutations and thus cause heart diseases. Thus, ICD is a hot a co-receptor) via a lipid-raft dependent pathway that activates spot for study of the structure and functional relationship of the signaling molecules Fyn and Abl kinases [144]. However, cardiomyocytes in health and diseases. To achieve this goal, in this Arf6-dependent entry was shown to require p42/p44 addition to the analyses of its components and organization, sev- MAPK activation triggered by CVB3 interaction with CAR eral new directions of studies have been emerged, such as i) the [175]. identification of signaling pathways related to ICD component proteins, ii) application of super-resolution microscopy and scan- ning ion-conductance microscopy to further refine the definition Conclusion and perspectives of the area composita, iii) identification of microRNAs involved in ARVC pathogenesis via targeting the ICD genes, and iv) The traditional classification divided the ICD structures into three particularly, the emerging of the new technology in deriving major complexes. Apparently, this idea is outdated. Recent stud- the iPSC cardiomyocytes using ARVC patient stem cells will ies demonstrated that the areas defining the junction groups be- provide various cell line models for the study [189]. In addition, come unclear because some junctional proteins were found not combining this approach with the CRISPR/Cas9 technique for restricted to distinct structures but exist almost completely in a genome editing, one can produce the required mutant hybrid and enforced structure [176, 177]. Thus, this ICD-specific cardiomyocytes based on the mutations found in the ICD gene hybrid junction has been termed area composite [178], which of patients. These cells, although only an in vitro model, could be initially included AJ and desmosome and later was expanded used to recapitulate pathological features in the context of the to include ion channels and GJs to distinct the term from adhe- patient’s genetic background and serve as a most useful platform sion junction [179]. This finding is supported by the following to study the pathogenesis of human heart diseases related to facts: Pkg is present in both AJ and desmosome [180]. Pkp-2 and mutation or destruction of ICD genes. Heart Fail Rev (2019) 24:115–132 127

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