Intercellular Connections in the Heart: The Intercalated Disc (Pathophysiology and Genetics of Cardiomyopathies) Part 3

Desmosomal cadherins

Desmosomal cadherins are a superfamily of Ca2+-dependent adhesion molecules, which form dimers to make up the core of desmosomal junctions (Dusek et al., 2007). Desmogleins and desmocollins, the two main types of desmosomal cadherins, possess several isoforms (4 and 3 respectively in humans, Green and Simpson, 2007; Lorimer et al., 1994; Schmelz et al., 1986) with desmoglein-2 and desmocollin-2 being the main isoforms expressed in mammalian cardiomyocytes (Garrod and Chidgey, 2008).

Desmoglein and Desmocollin: These classical cadherins are highly homologous; desmogleins and desmocollins share ~30% identity with each other and with other members of the cadherin family (Garrod et al., 2002). Much of their homology is found within their extracellular domains. They possess five extracellular domains or cadherin repeats of ~110 amino acids and are separated by Ca2+ binding motifs, which are necessary for dimerization (Fig 2B; Pokutta and Weis, 2007). A single-pass transmembrane domain and an intracellular anchoring segment follow the extracellular domains (Green and Simpson, 2007; Kowalczyk et al., 1999). Within their intracellular regions, desmogleins and desmocollins possess a cadherin-like sequence capable of binding catenins, or in the case of desmosomal cadherins, plakoglobin (Mathur et al., 1994).

Desmoglein and desmocollin differ significantly within their COOH-termini, however. In particular, the COOH-terminal region of desmoglein contains a proline-rich linker region, a series of short (~29 amino acids long) repeats and a glycine-rich terminal domain (Garrod and Chidgey, 2008; Holthofer et al., 2007), which likely mediates weak interactions with other desmosomal proteins (Kami et al., 2009). Conversely, alternative splicing within the COOH-terminus of desmocollin gives rise to two forms (Collins et al., 1991; Parker et al.,1991); the "b" or shorter form does not contain the traditional catenin-binding domain, however, the longer "a" form possesses a normal catenin-binding domain and has been shown to bind plakoglobin with high affinity (Troyanovsky et al., 1993).

Many studies suggest that both desmoglein and desmocollin are necessary for desmosomal formation (Getsios et al., 2004; Marcozzi et al., 1998; Tselepis et al., 1998). However, it is unclear if homophilic or heterophilic interactions maintain desmosomal adhesion. Although heterophilic complexes between desmoglein-2 and desmocollin-2 have been reported, it has been suggested that homophilic interactions between desmogleins mediate complex formation (Heupel et al., 2008; Syed et al., 2002; Waschke et al., 2005). Nonetheless, the importance of both desmoglein and desmocollin in cardiac function is further evidenced by the numerous mutations identified in their respective genes that lead to cardiomyopathies, mainly manifested as ARVC. Consistent with this, mice harbouring a mutation resulting in a truncated form of desmoglein-2 develop ARVC (Krusche et al., 2011), while a systemic knockout mouse model of desmoglein-2 is embryonic lethal (Eshkind et al., 2002).

Proteins of the catenin/armadillo family

Desmosomal cadherins form cytoplasmic connections with intermediate filaments in part through proteins of the armadillo family. Armadillo proteins include plakoglobin (also called y-catenin) and plakophilin, which are found at desmosomal structures (Cowin et al., 1986; Hatzfeld, 2005; Hatzfeld, 2007; Mertens et al., 1996; Mertens et al., 1999; Peifer et al.,1992), in addition to P-catenin, a-catenin and p120 catenin, which are mainly associated with adherens junctions (Hatzfeld, 2005; Hatzfeld, 2007). In addition to facilitating the anchoring of desmosomes to intermediate filaments, desmosomal armadillo proteins function in diverse signal transduction pathways.

Plakoglobin: Plakoglobin contains 12 arm repeats, which share 65% identity with the ones present in P-catenin, and are flanked by Pro-Lys-Gly rich NH2- and COOH-terminal domains (Fig. 2C; Garrod and Chidgey, 2008; Huber et al., 1997; Peifer et al., 1992). Mutation analysis suggested that plakoglobin interacts with desmosomal cadherins through its NH2-terminal domain as well as the arm repeats near its COOH-terminus (Chitaev et al., 1996; Wahl et al., 1996). Although the Pro-Lys-Gly motif interacts with both desmosomal and adherens junction cadherins, it has a higher affinity for desmoglein supporting plakoglobin’s mainly desmosomal localization (Chitaev et al., 1996; Choi et al., 2009). Moreover, through its central arm repeats plakoglobin interacts with desmoplakin, which in turn binds to intermediate filaments.

Plakophilin: Plakophilins undergo alternative splicing giving rise to four products, referred to as plakophilin 1-4; (reviewed in Bass-Zubek et al., 2009), with plakophilin-2 being the most prominent form in mammalian cardiomyocytes (Mertens et al., 1996). Plakophilins contain 9 arm repeats flanked by an NH2-terminal head and a short COOH-terminal region (Fig. 2C; Bass-Zubek et al., 2009). In addition, plakophilins 1-3 possess a flexible insertion between repeats 5 and 6, which introduces a major bend to their overall structure (Choi and Weis, 2005). Plakophilins bind to several desmosomal proteins through their NH2-terminal regions, including desmocollin, desmoplakin and plakoglobin as well as actin and the intermediate filament proteins keratin and desmin (Hofmann et al., 2000). Notably, plakophilin-2 also interacts with ankyrin-G at the ID, a sodium channel anchoring protein and with connexin-43 (Sato et al., 2011). Consequently, loss of plakophilin-2 leads to a decrease in the level of the a-subunit of the sodium channel (Nav1.5) at the membrane, which results in slow propagation of the action potential in cardiocytes (Sato et al., 2009). In addition to ankyrin-G, plakophilin-2 interacts with PKCa, which is necessary for phosphorylation and recruitment of desmoplakin to newly forming desmosomes in the developing heart and during repair of myocardial injury (reviewed in Garrod and Chidgey, 2008). Thus, through its multiple interactions, plakophilin-2 may serve as a scaffold to contribute to adhesion and signalling at the ID by facilitating the lateral interaction between desmosomes and adherens junctions (Kowalczyk et al., 1999).

The critical roles of both plakoglobin and plakophilin-2 in desmosomal assembly and maintenance is evidenced by the severe phenotypes that relevant transgenic mice models exhibit and the different forms of heart disease associated with mutations in their respective genes (please see Tables 1 and 2). Consistent with this, both plakoglobin and plakophilin-2 null mice show premature death during embryogenesis because of myocardial fragility (Bierkamp et al., 1996; Grossmann et al., 2004; Ruiz et al., 1996). Similarly, cardiac-specific knockout of plakoglobin results in progressive development of cardiac dysfunction (Li et al., 2011).

Plakins

Desmoplakin: Plakins are large multi-domain proteins that mediate the interaction of intermediate filaments (desmin in heart) with desmosomes. Desmoplakin, the main plakin protein expressed in heart, is characterized by a central a-helical coiled-coil rod domain, which is flanked by globular NH2- and COOH-termini (Fig. 2D; Franke et al., 1982). Through its coiled-coil region, desmoplakin has been suggested to form homodimers (Kowalczyk et al., 1994), while its NH2-terminal region binds to plakoglobins and plakophilins, targeting them to desmosomes (Bornslaeger et al., 1996; Bornslaeger et al., 2001; Holthofer et al., 2007; Kowalczyk et al., 1999). Its COOH-terminal tail is composed of three plakin-repeat domains and a Gly-Ser-Arg rich motif; both shown to mediate binding to desmin (Choi et al., 2002; Getsios et al., 2004). Interestingly, mice lacking desmoplakin exhibit embryonic lethality characterized by reduced number of desmosomes with residual structures separated from intermediate filaments (Gallicano et al., 1998). These results, along with the various desmoplakin mutations associated with human genetic disorders (please see below) support a strong role for desmoplakin in the assembly and interlinking of desmosomes to desmin intermediate filaments in cardiomyocytes.

	Major Proteins	References	Animal Models	Phenotype	References
	Connexin-43	Beyer et al., 1987	Systemic KO	Embryonic lethal	Reaume et al., 1995
			Cardiac Specific KO	Sudden cardiac death ~2 months	Gutstein et al., 2001b
	ZO-1	Giepmans et al., 1998; Toyofuku et al., 1998	Systemic KO	Embryonic lethal	Xu et al., 2008; Katsuno et al., 2008
	Caveolin	Schubert et al., 2002	Systemic KO	Development of DCM	Zhao et al., 2002
	Microtubule	Shaw et al., 2007	N/A	N/A	N/A
	N-Cadherin	Volk et al., 1984	Systemic KO	Embryonic lethal	Radice et al., 1997
			Cardiac specific KO	Sudden cardiac death ~2 months	Li et al., 2005; Kostetskii et al., 2005
			Dual heterozygote with connexin-43	Develop arythmias	Li et al., 2008
	P-catenin	Butz et al., 1995	Systemic KO	Embryonic lethal	Haegel et al., 1995
			Cardiac specific KO	Low survival rate	Piven et al., 2011
	a-catenin	Butz et al., 1995	Cardiac specific KO	Development of DCM	Piven et al., 2011; Sheikh et al., 2006
	P120 catenin	Aho et al., 1999	N/A	N/A	N/A
	Desmocollin-2	Lorimer et al., 1994	N/A	N/A	N/A
	Desmoglein-2	Schmelz et al., 1986	Transgenic lacking extracellular domains	Develop ARVC	Krusche et al., 2011
			Systemic KO	Embryonic lethal	Eshkind et al., 2002
	Plakoglobin	Peifer et al., 1992; Cowin et al., 1986	Systemic KO	Embryonic lethal	Bierkamp et al., 1996; Ruiz et al., 1996
			Cardiac Specific KO	Premature death due to cardiac dysfunction	Li et al., 2011
	Plakophilin-2	Mertens et al., 1996; Mertens et al., 1999	Systemic KO	Embryonic lethal	Grossmann et al., 2004
	Desmoplakin	Franke et al., 1982	Systemic KO	Embryonic lethal	Gallicano et al., 1998; Uzumcu et al., 2006
			Tetraploid rescue of systemic KO	Embryonic lethal	Gallicano et al., 2001
			Val30Met & Gln90Arg cardiac specific mutations	Embryonic lethal	Yang et al., 2006

Table 1. Listing of major proteins found at the ID and associated animal models with appropriate references; DCM: Dilated Cardiomyopathy, N/ A: not applicable, and KO: knock-out.

Table 2. Listing of mutations found in desmosomal genes that have been causally linked to the development of ARVC or variations of it; bp: base pair, IVS or AVSAS: denotes a splice site mutation, IVS: intervening sequence, AS: acceptor splice site, nt: nucleotide, ins: insertion.

ID proteins in human heart disease

Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a progressive disease characterized by loss of the right ventricular myocardium, and at advanced stages of the left ventricular myocardium as well, accompanied by fibro-fatty tissue infiltration and replacement. Its clinical manifestations include ventricular arrhythmias, syncope, heart failure and sudden cardiac death (Delmar and McKenna, 2010; Estigoy et al., 2009; Lombardi and Marian, 2011). ARVC has an estimated prevalence of 1 in 5,000 (Sen-Chowdhry et al., 2010), although in some regions (e.g. northern Italy) it reaches 1 in 2,000 (Thiene et al., 2007). Genetic studies have indicated that ~50% of the diagnosed ARVC cases are familial, with an autosomal dominant inheritance (Marcus et al., 1982). Accordingly, a number of mutations have been identified in the genes that encode cardiac desmosomal proteins, and thus ARVC is also referred to as "a disease of the desmosome" (Li and Radice, 2010). These mutations not only affect the number, structural integrity and proper localization of desmosomes, but also of gap junctions, resulting in impaired intercellular conductance and thus development of arrhythmias. To date, five desmosomal genes have been identified that carry inherited mutations causing different variations of ARVC, including: plakophilin-2, desmocollin-2, desmoglein-2, plakoglobin and desmoplakin. Table 2 includes a comprehensive list of mutations identified to date in these five desmosomal genes; for updated listing please refer to: http://www.ncbi.nlm.nih.gov/omim. More than 70% of the identified desmosomal mutations associated with the development of familial ARVC are present in the gene encoding plakophilin-2 (Gerull et al., 2004; Sen-Chowdhry et al., 2010; van Tintelen et al., 2007). These account for ~20% of diagnosed ARVC cases, while mutations in the genes encoding desmocollin-2 (Simpson et al., 2009; Syrris et al., 2006) and desmoglein-2 (Awad et al., 2006; Posch et al., 2008; Syrris et al., 2006) account for ~10-15% of cases each (Lombardi and Marian, 2011; Pilichou et al., 2006). Plakoglobin was the first desmosomal protein to be causally associated with a cardiocutaneous subtype of ARVC, known as Naxos disease, which was first characterized by Protonotarios et al. (Protonotarios et al., 1986). Genetic studies of patients from the Greek island Naxos, where the syndrome took its name from, revealed a homozygous two-base-pairs deletion (2157-2158delGT) in the gene encoding plakoglobin that was inherited in an autosomal recessive manner (McKoy et al., 2000). In addition to developing ARVC, these individuals also suffered from palmoplantar keratoderma and woolly hair. Recently though, a variation of the Naxos syndrome was diagnosed in a German family that carried a dominantly inherited mutation in the plakoglobin gene (Ser39Lys40insSer) that caused ARVC without the accompanying cutaneous abnormalities (Asimaki et al., 2007). Importantly, the reduced expression or complete absence of plakoglobin from the ID of ARVC patients is a consistent feature, making it a valuable marker for its diagnosis, which still remains problematic with many cases being un- or misdiagnosed.

Mutations in the gene encoding desmoplakin have been identified as the underlying cause of a variation of Naxos disease, referred to as Carvajal syndrome that is also characterized by woolly hair, palmoplantar keratoderma and cardiac disease (Kaplan et al., 2004a; Kaplan et al., 2004b; Norman et al., 2005; Rampazzo et al., 2002; Saffitz, 2009; Yang et al., 2006; Bauce et al., 2010; Bolling et al., 2010; Norgett et al., 2000; Uzumcu et al., 2006). Notably, cardiac disease is presented as a generalized hypertrophy and dilation, involving both the right and left ventricles, and accompanied by focal ventricular aneurysms without any apparent fibro-fatty tissue replacement (Kaplan et al., 2004a; Yang et al., 2006). A major feature of the Carvajal syndrome is the virtual absence of desmoplakin in the affected hearts, indicating that the missense or nonsense mutations identified result in truncated and/or unstable forms of the protein (Norman et al., 2005; Rampazzo et al., 2002).

Alterations in the amounts, localization and functional properties of desmosomal proteins not only affect intercellular adhesion, but also promote remodelling of gap junctions by leading to abnormal expression and distribution of gap junctional proteins, and primarily connexin-43, which in turn induces defects in the electrochemical coupling of neighbouring cardiocytes and leads to the development of severe arrhythmias (Kaplan et al., 2004a; Pieperhoff et al., 2008; Saffitz, 2009). On the contrary, changes in gap junctions do not affect the structural integrity or proper function of desmosomes and adherens junctions, and thus mechanical coupling of adjacent cardiocytes is not disrupted (Delmar and McKenna, 2010; Li and Radice, 2010; Noorman et al., 2009).

A number of mutations have also been identified in the gene encoding connexin-43, which are associated with the development of oculodentodigital dysplasia (ODDD) that is frequently accompanied by hair and skin defects, too (Kelly et al., 2006). Some of these mutations have been further linked to the development of cardiac disturbances. In such patients, the expression levels of connexin-43, and thus the number of gap junctions, are moderately decreased (Manias et al., 2008); however, cardiac conduction is not affected. Thus, sole mutations in the gene encoding connexin-43 cannot be the primary inducers of electrical or mechanical defects underlying arrhythmogenesis. Interestingly, neither loss-nor gain-of-function mutations have been identified in proteins of adherens junctions that are causally associated with the development of cardiac disease. A plausible explanation is that dysfunctional adherens junctions may be detrimental to the developing myocardium and thus may result in embryonic lethality. Consistent with this, a constitutive null model of N-cadherin was embryonic lethal, while a developmental and cardiac tissue specific model developed dilated cardiomyopathy and died 2 months following excision of the gene, due to mechanical and electrical abnormalities (Li et al., 2005; Radice et al., 1997); for review of available animal models of N-cadherin and their phenotypic characterization, please refer to (Li et al., 2006).

Concluding remarks: The intercalated disc is a single functional unit

Although traditionally depicted as a composition of three separate units, data from the last decade suggest that the ID of cardiomyocytes is in fact a single functional unit. Several studies have begun to describe area composita as a hybrid between proteins of adherens junctions and desmosomes that form a single anchoring unit (Delmar, 2004; Franke et al., 2006; Pieperhoff and Franke, 2007; Saffitz, 2005). Consistent with this, plakophilin-2 and desmoglein, which typically localize to desmosomes, interact with P- or a-catenin and p120 catenin, respectively, present in adherens junctions (Chen et al., 2002; Goossens et al., 2007). Similarly, molecular linkages between desmosomes and gap junctions have also been identified (Rohr, 2007; Saffitz, 2005). As such, desmocollin-2 directly interacts with connexin-43 (Gehmlich et al., 2011). Taken together, these studies clearly suggest that there is a three-way exchange and cross-talk of junctional proteins, supporting the idea of the ID being a single functional unit.

During the last decade, there have been significant advancements concerning the structural composition of the ID. A plethora of new proteins has been identified as integral or peripheral components of the ID that directly or indirectly contributes to the mechanical and electrical coupling of neighbouring cardiocytes. The challenge of the future lies in the characterization of the precise roles that these proteins play to ensure the synchronous contraction of the myocardium. A combination of sophisticated molecular, genetic and cellular approaches will be needed to address this unequivocally important question.