Cell Communication & Adhesion, 21: 149–159, 2014 © 2014 Informa Healthcare USA, Inc. ISSN: 1541-9061 print / 1543-5180 online DOI: 10.3109/15419061.2014.905928

REVIEW ARTICLE

Cell Junctions in the Specialized Conduction System of the Heart Valeria Mezzano1*, Jason Pellman2*, and Farah Sheikh2 1

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Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, New York and 2 Department of Medicine, University of California, San Diego, CA, USA

Abstract Anchoring cell junctions are integral in maintaining electro-mechanical coupling of ventricular working cardiomyocytes; however, their role in cardiomyocytes of the cardiac conduction system (CCS) remains less clear. Recent studies in genetic mouse models and humans highlight the appearance of these cell junctions alongside gap junctions in the CCS and also show that defects in these structures and their components are associated with conduction impairments in the CCS. Here we outline current evidence supporting an integral relationship between anchoring and gap junctions in the CCS. Specifically we focus on (1) molecular and ultrastructural evidence for cell–cell junctions in specialized cardiomyocytes of the CCS, (2) genetic mouse models specifically targeting cell–cell junction components in the heart which exhibit CCS conduction defects and (3) human clinical studies from patients with cell–cell junction-based diseases that exhibit CCS electrophysiological defects. Keywords: cell junction, cardiac conduction system, desmosome, fascia adherens junction, gap junction, intercalated disc, cardiac muscle, heart, heart disease, anchoring junction

STRUCTURE AND FUNCTION OF THE SPECIALIZED CONDUCTION SYSTEM

them in terms of their cytoarchitecture and electrophysiological characteristics. In this review we will focus on the architectural components found at the cell–cell junctions in the CCS and discuss the similarities and differences between these and the better understood junctions within working myocytes as well as highlight genetic mouse models and human data that provide functional relationships to CCS electrophysiology. The PC is located at the junction between the right atrium and superior caval vein (Chandler et al., 2011; Mangoni & Nargeot, 2008). Shaped like a comma with its tail bending inferiorly along the crista terminalis, it is composed of a network of several thousand cells that are smaller in size than working atrial and ventricular myocytes with various shapes including some with prolongations capable of forming extensive connections with many other cardiomyocytes (Shimada et al., 2004). Like working cardiomyocytes, pacemaker cells have abundant mitochondria and glycogen but their sarcomeres are sparse and generally their contractile apparatus is less abundant. The signal that arises from the PC depolarizes both atria, through preferential conduction pathways (Fedorov et al., 2012; Stiles et al., 2010), and is then relayed to the AVN. The AVN is a triangular structure located at the triangle of Koch. Anatomical boundaries described for the human AVN include the tendon of Todaro, the coronary sinus ostium, and the hinge of the septal leaflet of the tricuspid valve (Anderson et al., 2009). The conduction path from the AVN into the ventricular myocardium

The mammalian cardiac conduction system (CCS) coordinates the pumping function of the heart by producing and distributing the initial depolarization signal in a specific spatiotemporal pattern. This feat is accomplished by a very well-defined network of specialized cardiomyocytes that has the ability to generate and propagate action potentials in a way that will lead to atrial contraction and subsequent ventricular contraction (Boyett, 2009). This network is formed by (i) sinoatrial nodal cells that give rise to the initial depolarization and which are referred to as the pacemaker complex (PC; also known as sinoatrial node [SAN]), (ii) intra-atrial conduction pathway myocytes that will carry the impulse into the atrioventricular node (AVN), which provides the temporal delay for the activation of the ventricles, and (iii) the His-Purkinje system cardiomyocytes that distributes the depolarization signal throughout the ventricles. Cardiomyocytes within the specialized CCS share some features similar to working myocytes found in the atrial and ventricular myocardium, but also differ from Received 3 March 2014; accepted 16 March 2014. *Both authors contributed equally to this work. Address correspondence to Farah Sheikh, Department of Medicine (Cardiology Division), University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0613C, USA. Tel: ⫹(858) 246-0754. Fax: ⫹(858) 822-1355. E-mail: [email protected]

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involves the bundle of His (or penetrating bundle) that lies over the muscular part of the interventricular septum toward the left outflow tract (Anderson et al., 2009). From there it divides into the right and left bundle branches at either side of the septum. This description is a simplified version of the conduction pathways distal to the AVN as it is increasingly clear that specialized conduction structures also exit inferiorly from the node forming rings around the mitral and tricuspid valves and a retroaortic node (Yanni et al., 2009; Nikolaidou et al., 2012). These structures have not been as extensively described as the other conduction system compartments, but are slowly becoming better understood from morphological and electrophysiological perspectives. The purkinje network has been elegantly identified in different animal model systems through specific immunostaining techniques and/or genetic lineage tracing tools (Rentschler et al., 2001; Pallante et al., 2010; Atkinson et al., 2011). It forms a net of purkinje fibers (PFs) on the endocardial surface of the septum and parietal walls and distributes into free running PF and a terminal PF that will transmit the action potential to the ventricular muscle (Atkinson et al., 2011).

LOCATION OF CELL–CELL JUNCTION COMPONENTS IN THE CCS The cells that form the CCS share some similar features with working myocytes, while they differ in others. Identifying these structures has relied upon anatomical and histological analyses, functional electrophysiological mapping, immunolocalization of specific proteins found in the CCS (most commonly ion channels) as well as characterization of specific genetically engineered mouse models (reviewed below). Thus, the identification of specialized cardiomyocytes of the CCS has emerged from both histological analyses and functional studies. In the same manner, evidence for specific cell–cell junctions and their component proteins in these cells has been achieved through ultrastructural analyses (i.e., electron microscopy) as well as molecular and cellular identification of specific proteins through gene expression studies. As the genetic markers for the CCS became more precise, so has the understanding of the intercellular structures present in these cells. The junction between the ends of cylindrical myocytes of the ventricular and atrial working myocardium has been termed the intercalated disc (ICD). Through hematoxilin and eosin staining methods, the ICD appears as a thick eosinophilic band in working cardiomyocytes. Higher resolution imaging of this band highlights a composition consisting of various “steps and risers”, which have been elegantly shown by Shimada et al. (2004) through scanning electron micrographs and specifically “characterized by the presence of a large number of finger-like microprojections”. However, these steps have not been observed

in similar preparations obtained from pacemaker and AVN cardiac cells. Transmission electron microscopy studies highlighted that these microprojections form part of the actin microfilaments that dock at the fascia adherens junction (FA; also known as adherens junction) within the ICD of ventricular cardiomyocytes. Meanwhile, the “risers” which lack microprojections were thought to be sites of gap junction plaque localization. Interestingly, both the “steps” and “risers” were associated with the presence of desmosomal structures. These three main multiprotein structures that link one cell to the next in working myocardium are also found in the CCS but in a slightly altered form/localization given the different cellular architecture of the CCS. Using the ICD of the ventricular cardiomyocyte as a starting point and a simplified understanding of the cell–cell junctions found between cardiomyocytes, a parallel of what structures have been shown at the CCS can be made (Figure 1). Desmosomes Desmosomes link the intermediate filaments of two neighboring myocytes through a complex of proteins that have both mechanical and signaling properties (Delva et al., 2009). Evidence for the presence of specific desmosomal structures in the CCS is clear, and has been obtained through: (i) electron microscopy of desmosomal structures found at the SAN (Shimada et al., 2004; Saffitz et al., 1997) and AVN (Vassall-Adams, 1983; Shimada et al., 2004) and (ii) immunohistochemical evidence of specific desmosomal components, such as desmoplakin, a central component of the desmosome (Dobrzynski et al., 2000) as well as plakoglobin (Lim et al., 2008) in the SAN and AVN. Gap junctions Gap junctions (nexus) provide a platform for small molecule exchange between neighboring cells. In working cardiomyocytes they tend to localize to the “risers” of the ICD. However, in the CCS there appear to be no “risers” and therefore their localization is different. In the SAN, gap junctions are found at lateral and terminal ends of cells and are smaller than the plaques found in ventricular muscle (Saffitz et al., 1997). Furthermore, specific proteins of the gap junction (connexins) that form these junctions in the CCS differ from those found in working cardiomyocytes. For example in the SAN the following gap junction proteins have been reported: Connexin (Cx)30, Cx30.2 (in mouse) (Gros et al., 2010; Kreuzberg et al., 2006) and Cx45 (Coppen et al., 1999a, 2003). Likewise in the AVN both Cx30.2 (Kreuzberg et al., 2006; Kreuzberg et al., 2005) and Cx45 (Severs et al., 2008; Coppen et al., 1999b) are specifically expressed. Interestingly conductance of these particular connexins differ from Cx43 (the most abundant ventricular connexin protein). It has been proposed, based on in vivo experiments with mice, that the lower conductance

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CELL JUNCTIONS IN CARDIAC CONDUCTION SYSTEM

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Figure 1. Schemata of cell–cell junction structures (S) and associated molecular components found in the cardiac conduction system.

(Kreuzberg et al., 2005) of gap junctions found at this location (e.g., Cx30.2) serves to decelerate the impulse propagation at the AVN (Kreuzberg et al., 2006). Structurally, as in the SAN, gap junctions in the AVN are very small when compared to those found in working cardiomyocytes (Shimada et al., 2004). As part of the ventricular conduction system, which encompasses the His bundle, bundle branches and purkinje network, the main gap junction proteins present include Cx40 (Miquerol et al., 2004, 2011; Kanter et al., 1995) and Cx45 (Coppen et al., 1999b). Fascia adherens junctions Like desmosomes, FA connect the cytoskeletal elements (in this case microfilaments, specifically actin) of two neighboring myocytes through transmembrane cadherins. In working cardiomyocytes, they are found at the ICD linking together the microprojections described by Shimada et al. (2004). Unlike desmosomes, there is no central protein that mediates binding between actin and the cadherins but a complex of proteins that also have signaling properties (β-Catenin, ZO-1, plakophilin 2, vinculin) (Hatzfeld et al., 2014; Koetsier et al., 2014; Xu et al., 2012; Holle et al., 2013), which are also present at these anchoring sites. Although proteins that form part of the FA are found in the AVN, a typical ICD has been difficult to recognize within this tissue, which may be due to the fact that the nodal cells have ramifications, fewer microprojections and fewer sarcomeres (Shimada et al., 2004).

Composite junctions (Area composita) Evidence for composite junctions in the CCS is provided by electron microscopy combined with immunodetection of specific proteins in the purkinje cells of bovine tissue. Studies demonstrated clear immunolabeling of desmoplakin in these cells accompanied by the expression of the desmosomal cadherin, desmoglein-2, as well as the FA proteins, N-cadherin and β-catenin (Pieperhoff et al., 2010). Unlike working cardiomyocytes, these junctions reside at the longitudinal borders of purkinje cells providing mechanical stabilization to the ventricular conduction system. CCS PHENOTYPES IN ANIMAL MODELS WITH UNDERLYING DEFECTS IN CELL–CELL JUNCTIONAL COMPONENTS Although the direct functional role for cell–cell junction proteins in the CCS has not been fully established, genetic mouse models targeting loss of these junctional components have paved the way toward demonstrating their molecular, architectural, and functional role in CCS structures ranging from the SAN to the PF network (Table 1). A caveat to note is that nearly all cardiac musclespecific knockout mouse models targeting cell-cell junction proteins rely on conventional knockout or Cre-mediated knockout strategies under the control of a cardiac-specific Cre (e.g., αMHC-Cre and MLC2v-Cre) that also targets working cardiomyocytes rather than

Desmoplakin (α-MHC-Cre; heterozygous)

Plakophilin 2 (global heterozygote)

Plakoglobin (global heterozygote) Desmosome Desmoglein 2-N271S mutation (cardiac- specific transgenic)

CAR (α-MHC-mERCre-mER)* FA junction and desmosome Plakoglobin/β-catenin double knockout (αMHC-mER-CremER) Plakoglobin (αMHC-Cre)

CAR (αMHC-Cre)

mXin-alpha (global)

N-cadherin/Connexin 43 (global double heterozygotes)* Vinculin (global heterozygous)* Vinculin (MLC2v-Cre)

FA junction N-cadherin (αMHC-mER-CREmER)

Genetic mouse model

Low amplitude of QRS complex, prolonged PR interval and spontaneous non-sustained ventricular tachycardia, spontaneous ventricular ectopic beats Ventricular tachycardia Premature ventricular beats, monomorphic and polymorphic ventricular tachycardia and atrial arrhythmias Ventricular conduction slowing and susceptibility to induced Ventricular arrhythmias Baseline prolongation of P-wave duration. Challenge with Nav1.5 channel blocker, flecainide, causes prolonged PR, QRS and QTc intervals plus second degree AV block and ventricular arrhythmias Spontaneous ventricular ectopic beats, ventricular couplets as well as susceptibility to spontaneous and induced ventricular tachycardia

Absent desmosomal structures

Young age (low, high expressors): No reported effects on ICD Old age (low expressors): Ultrastructural ICD defects Ultrastructural defects at ICD, including loss of desmosomes

Not examined

No reported effects on ICD

Prolonged PR and QRS intervals, lethal spontaneous ventricular arrhythmias, susceptibility to induced ventricular fibrillation

Prolonged PR interval, 1st, 2nd and 3rd degree AV block, reduced AV conduction capacity, sinus node dysfunction

Complete or 1st degree AV block, prolonged PR interval

(Continued )

Garcia-Gras et al. (2006)

Cerrone et al. (2012)

Pilichou et al. (2009), Rizzo et al. (2012)

Kirchhoff et al. (2006)

Li et al. (2011)

Swope et al. (2012)

Lisewski et al. (2008)

Lim et al. (2008)

Gustafson-Wagner et al. (2007)

Zemljic-Harpf et al. (2004) Zemljic-Harpf et al. (2007)

Widened QRS complex AV block, polymorphic ventricular tachycardia Prolonged P-wave duration and QT interval

Li et al. (2008)

Kostetskii et al. (2005), Li et al. (2005)

References

Longer PR and QRS interval, QRS amplitude lower, P-wave amplitude higher, longer HV interval, susceptibility to induced atrial and ventricular arrhythmias Susceptibility to induced ventricular arrhythmias

Cardiac electrophysiological abnormalities

Absent ICD structures

Molecular reduction of specific FA and GJ components at ICD Myofibril connection to ICD is affected Ultrastructural defects at ICD, including connection to myofibrils Ultrastructural and molecular (specific FA, D, GJ components) defects at ICD, including fewer gap junctions Ultrastructural and molecular (specific FA and GJ components) defects at ICD Molecular defects (specific components of GJ) at ICD

Absent ICD structures

Effects on cell–cell junction structure and components

Table 1. Summary of genetic mouse models harboring defects in cardiac muscle cell–cell junction components that exhibit electrophysiological defects.

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Prolonged PQ and AH intervals Prolonged PQ and AH intervals, susceptible to mechanically induced AV block Spontaneous ventricular tachycardia, reduced ventricular conduction velocity Ectopic ventricular beats or sustained ventricular tachycardia (VT), increased anisotropic ratios Spontaneous ventricular tachycardia, reduced ventricular conduction velocity Prolonged QRS and QT intervals, reduced HV intervals, inducible atrial tachycardia and fibrillation Prolonged QRS interval and subsequent age dependent decrease, inducible ventricular tachycardia, reduced conduction velocity

Not examined Not examined

Not examined Not examined Not examined

Connexin 43 (Cx43-Cre-ER(T))⫹

Connexin 43 (chimeric knockout) Connexin 43 G60S heterozygous mutant⫹ Connexin 43 (aMHC-Cre selectively bred for longevity)*

CAR: Coxsackievirus-adenovirus receptor ICD: Intercalated disc GJ: Gap junction D: Desmosomes FA: Fascia adherens *not associated with cardiomyopathy. ⫹ Not examined for cardiomyopathy.

Not examined

Not examined

Prolonged QRS interval, monomorphic and polymorphic ventricular arrhythmias

1st degree AV block, bundle branch block, prolonged P-wave, PR, QRS, QT, AH, and HV intervals

Prolonged QRS interval, spontaneous ectopic premature ventricular contractions in anaesthetized mice, spontaneous ectopic multifocal premature beats in isolated hearts and right bundle branch block

Cardiac electrophysiological abnormalities

Not examined

Not examined

Ultrastructural and molecular (specific D and GJ components) defects at ICD

Effects on cell–cell junction structure and components

Connexin 43 premature stop (conditional Cx43D378stop via aMHC-mER-Cre-mER)⫹ Connexin 45 (αMHC-CreER(T2))⫹ Connexin 45 (αMHC-CreER(T2))/Connexin 30.2 (global) double knockout⫹ Connexin 43 (αMHC-Cre)⫹

Gap junction Connexin 40 (global)⫹

Desmoplakin (MLC2v-Cre)

Genetic mouse model

Table 1. (Continued )

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Danik et al. (2004)

Tuomi et al. (2011)

Gutstein et al. (2001b)

van Rijen et al. (2004)

Gutstein et al. (2001a)

Frank et al. (2012)

Frank et al. (2012)

Simon et al. (1998), Kirchhoff et al. (1998), Bevilacqua et al. (2000), Tamaddon et al. (2000), Hagendorff et al. (1999), Verheule et al. (1999), VanderBrink et al. (2000), Zhu et al. (2005), van Rijen et al. (2001), Bagwe et al. (2005), Leaf et al. (2008) Lübkemeier et al. (2013)

Lyon et al. (2014)

References

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a CCS-specific Cre that only targets the CCS. Therefore, in this case, the intrinsic effects on the CCS may be masked or augmented by functional effects on working cardiomyocytes.

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Models with SAN dysfunction Evidence for a role for cell–cell junction proteins in the SAN has been observed in very few genetic mouse models; however, this could be confounded by the difficulty in analyzing SAN dysfunction, which is not possible without rigorous and careful cardiac electrophysiological studies. SAN dysfunction presents as an inappropriate sinus bradycardia, sinus arrest/pause, and sinus node exit block (Rubenstein et al., 1972). In a genetic mouse model targeting loss of Coxsackievirus-adenovirus receptor (CAR) via αMHC-mER-Cre-mER, sinus node dysfunction was observed as sinus node tachycardia and bradycardia, which was also coincident with loss of Cx45 (Lisewski et al., 2008), a gap junction protein predominantly expressed in the SAN and AVN (Coppen et al., 1999a, 2003). Cx40 function has also been implicated in SAN initiation through mouse models. Global loss of Cx40 leads to longer sinus node recovery times and longer P-waves (Hagendorff et al., 1999; Verheule et al., 1999; Bagwe et al., 2005). At embryonic stages (E15.5), global loss of Cx40 leads to ectopic SAN impulse initiation (Leaf et al., 2008). However, this dysfunction was not present in adult Cx40 knockout mice (Leaf et al., 2008). Models with AVN dysfunction AVN dysfunction has been observed in various genetic mouse models targeting loss of cell–cell junction components. AVN dysfunction is most obviously observed as an AV block upon ECG analysis, which includes 1st degree (prolonged PR interval), 2nd degree (P-waves without accompanying QRS complex), and 3rd degree or complete (QRS completely independent of P-waves) heart block (Dobrzynski et al., 2013). AVN function can also be determined by its conductance speed, measured by an AH interval or the speed of depolarization from first rapid atrial deflection to the His bundle deflection (Dobrzynski et al., 2013). Various FA knockout mouse models have been shown to display evidence of AVN dysfunction. These include genetic mouse models with targeted loss of N-Cadherin (αMHC-mER-Cre-mER: prolonged PR interval) (Kostetskii et al., 2005; Li et al., 2005), vinculin (MLC2v-Cre: AV block) (Zemljic-Harpf et al., 2007), and CAR (αMHC-Cre; 1st or 3rd degree AV block and αMHC-mER- Cre-mER: 1st, 2nd, and 3rd degree AV block) (Lim et al., 2008; Lisewski et al., 2008). All genetic mouse models also displayed ultrastructural defects at the ICD of working cardiomyocytes; however, no specific studies analyzing the ICD in the AVN were performed. Altogether these data suggest that FA proteins play a direct or indirect role in AVN function.

Evidence of AV node dysfunction has also been observed in mice lacking specific desmosomal proteins in the heart. Global heterozygous loss of plakophilin-2 in mice lead to flecainide challenge-induced prolonged PR intervals and 2nd degree AV block, which were coincident with ultrastructural defects at the ICD that include sporadic and absent desmosomes (Cerrone et al., 2012). Specific knockout mice with targeted loss of gap junction proteins also displayed features of AVN dysfunction, which support the expression of multiple connexins in the AVN. For example, connexin 40 global knockout mice exhibited 1st degree AV block (Simon et al., 1998; Kirchhoff et al., 1998; Bevilacqua et al., 2000; Tamaddon et al., 2000; VanderBrink et al., 2000; Zhu et al., 2005), while targeted loss of connexin 45 in mice via αMHCCre-ER (T2) lead to a prolonged PQ and AH intervals suggestive of AVN dysfunction (Frank et al., 2012). Models with His-Purkinje system dysfunction His-Purkinje system dysfunction typically presents as arrhythmias (e.g., ventricular tachycardia or torsade de pointes) or as a bundle branch block. These can be determined via ECG analysis as arrhythmias and widened QRS intervals or via electrophysiology techniques those directly measure stress/exercise or catecholamineinduced arrhythmias and conduction velocity through His bundles (HV interval) or ventricles (Dobrzynski et al., 2013). Mice lacking components of the FA in the heart also exhibited various electrophysiological phenotypes characteristic of His-Purkinje dysfunction. In mice lacking N-cadherin in the adult heart via αMHC-mER-Cre-mER displayed lower QRS amplitude, longer HV interval, and a susceptibility to induced ventricular arrhythmias (Kostetskii et al., 2005; Li et al., 2005). Susceptibility to induced ventricular arrhythmias is also observed in mice with heterozygous global loss of N-Cadherin and connexin 43 (Li et al., 2008), supporting a role for FA function in ventricular conduction. Mice harboring global heterozygous loss of vinculin also displayed widened QRS complex (Zemljic-Harpf et al., 2004). This electrophysiological phenotype was exacerbated in mice harboring complete cardiac-specific loss of vinculin using MLC2v-Cre, which exhibited polymorphic ventricular tachycardia and sudden death (Zemljic-Harpf et al., 2007). The more recently identified FA protein mXinalpha may also have a role in the His-Purkinje system as global loss of mXinalpha lead to longer QT intervals in addition to its defects on atrial conduction (GustafsonWagner et al., 2007). Mice lacking plakoglobin, which is found at both the FA and desmosome, also displayed features of His-Purkinje dysfunction. In plakoglobin loss-offunction mouse models (cardiac-specific plakoglobin knockout mice using αMHC-Cre and global plakoglobin heterozygous knockout), mice were shown to display low amplitude QRS complex, spontaneous ventricular

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CELL JUNCTIONS IN CARDIAC CONDUCTION SYSTEM tachycardia, and spontaneous ventricular ectopic beats (Kirchhof et al., 2006; Li et al., 2011; Swope et al., 2012). ICD defects were shown to be present in cardiacspecific plakoglobin knockout mice but not in global plakoglobin heterozygous knockout mice. Loss and/or mutations in desmosomal components have also been shown to lead to His-Purkinje associated electrophysiological phenotypes in mice. Global heterozygous loss of plakophilin-2 in mice lead to HisPurkinje system defects including flecainide challengeinduced prolonged QRS and QTc intervals as well as ventricular arrhythmias (Cerrone et al., 2012). Several studies targeting loss of desmoplakin in the mouse heart demonstrated profound defects in the His-Purkinje system conduction. Cardiac-specific heterozygous loss of desmoplakin in mice using the αMHC-Cre lead to mice harboring spontaneous ventricular ectopic beats, ventricular couplets as well as susceptibility to spontaneous and induced ventricular tachycardia (Garcia-Gras et al., 2006). Furthermore, cardiac-specific homozygous desmoplakin knockout mice generated using MLC2v-Cre displayed severe electrophysiological defects including prolonged QRS interval, spontaneous ectopic premature ventricular contractions in anaesthetized mice, and right bundle branch block (Lyon et al., 2014). Mouse models harboring human desmosomal gene mutations associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) also lead to electrophysiological features suggestive of His-Purkinje system dysfunction. Cardiac-specific overexpression of a human desmoglein 2 mutation (N271S) in young mice lead to premature ventricular beats as well as monomorphic and polymorphic ventricular tachycardia prior to changes in ICD structure (Pilichou et al., 2009). ICD structural changes were also found in older mice, which also displayed ventricular conduction slowing as well as susceptibility to induced ventricular arrhythmias (Rizzo et al., 2012). Examination of ventricular cardiomyocytes from mice lacking plakophilin-2, desmoplakin, or expressing the human desmoglein 2 mutant revealed ultrastructural defects at the ICD. Hearts from cardiac-specific desmoplakin homozygous knockout mice (via MLC2v-Cre) also revealed specific gap junction defects that may precede overt structural defects to the working myocardium (Lyon et al., 2014). Altogether these studies suggest that there may be a functional connection between the desmosome and His-Purkinje system. Coincident with the robust gap junction presence in the His-Purkinje network, loss of specific gap junction proteins also lead to various electrophysiological defects in heart function associated with His-Purkinje dysfunction. Several studies have shown that global loss of Cx40 in mice leads to His-Purkinje dysfunction (in addition to AVN function) with electrophysiological defects including bundle branch block and prolonged HV intervals (Simon et al., 1998; Bevilacqua et al., 2000; Tamaddon et al., 2000; VanderBrink et al., 2000; van Rijen et al., 2001). Cardiacspecific loss of Cx45 via αMHC-Cre-ER(T2) also lead to

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prolonged AH intervals suggestive of His bundle conduction slowing (Frank et al., 2012). Loss of Cx43 via various knockout methods typically leads to ventricular conduction dysfunction including prolonged QRS, ventricular arrhythmias, reduced ventricular conduction velocity, and increased anisotropic ratios which may also reflect its expression in the His-Purkinje system (Lübkemeier et al., 2013; Gutstein et al., 2001a; van Rijen et al., 2004; Gutstein et al., 2001b; Danik et al., 2004). However, the G60S mutation in Cx43 displays similar ventricular conduction defects along with reduced HV interval (Tuomi et al., 2011), suggesting that this mutation may impact conductivity within the His bundle. CCS DEFECTS FOUND IN HUMAN PATIENTS WITH UNDERLYING DEFECTS IN CELL–CELL JUNCTIONAL COMPONENTS A number of studies in patients harboring cardiac diseases associated with defects/mutations in cell–cell junction genes have revealed underlying CCS electrophysiological defects (Table 2). The majority of evidence comes from studies performed on patients harboring the cardiac muscle disease, ARVC, which is primarily thought to be caused by pathogenic mutations/defects in genes of the desmosomal cell–cell junction (Sheikh et al., 2009); however, studies have also revealed that patients harboring gap junction mutations also present with specific electrophysiological abnormalities linked to the CCS. Human cell–cell junction diseases with SAN dysfunction Patients with ARVC do not classically present with SAN dysfunction and it is not a diagnostic criterion for ARVC. However, a growing number of case studies have been published linking sick sinus syndrome and ARVC (Nogami et al., 1990; Takemura et al., 2008; Balderramo & Caeiro, 2004; Morady et al., 1984). The desmosomalbased etiology of ARVC (Sheikh et al., 2009) as well as structural and molecular evidence of desmosomes in the SAN (Shimada et al., 2004; Saffitz et al., 1997), altogether suggest a previously uncharacterized role for desmosomes in the SAN, though mouse models targeting desmosomal components have yet to directly address this potential role. Human cell–cell junction diseases with AVN dysfunction AVN dysfunction has not been typically associated with human mutations in cell–cell junction genes, though there has been a single case study in a patient with ARVC that exhibited AVN dysfunction when specifically measured (Nogami et al., 1990). Whether the weak association is due to a lack of a role for these proteins in the AVN or masking of the role by other more obvious

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Table 2. Summary of studies highlighting human cardiomyopathies associated with defects/mutations in cell–cell junction components and their influence on CCS electrophysiological defects.

Human disease

Mutations in or effects on cell–cell junction structure and components

Diseases with mutations in desmosomal genes Brugada Syndrome PKP2 Mutations ARVC ARVC

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ARVC

ARVC ARVC

ARVC ARVC ARVC Diseases with mutations in gap junction genes Familial conduction Potential Connexin 40 system disease mutation Progressive familial heart Connexin 40 mutation block type I (PFHBI)

Cardiac electrophysiological abnormalities

ST-segment abnormalities in leads V1–V3 on ECG and ventricular arrhythmias SAN, right atrium, and AVN conduction defects Sick sinus syndrome, premature ventricular contractions Sick sinus syndrome

Decreased QRS amplitude, inverted T-waves Ventricular tachycardia, late potential on signal averaged ECGs, terminal activation duration of QRS ⱖ 55 ms, inverted T-waves Epsilon waves, inverted T-waves, terminal activation delay, late potentials Ventricular tachycardia, inverted T-waves Prolonged terminal activation duration, inverted T-waves Fixed conduction disease or intermittent conduction disease (abnormal rhythm by Holter monitoring) Conduction disturbance in His-Purkinje system

cardiac defects in humans is still unclear. Because various genetic mouse model studies have revealed AVN roles for numerous cell–cell junction proteins, these studies suggest further exploration for a role for these proteins in the AVN of the human heart. Human cell–cell junction diseases with His-Purkinje system dysfunction ARVC is a prime example of a cardiac disease associated with mutations/defects in desmosomal cell–cell junction components leading to defects in cardiac conduction, which include specific defects in the HisPurkinje system (Zusterzeel et al., 2013; Quarta et al., 2011; Bae et al., 2013; Bao et al., 2013; Cox et al., 2011) (Table 2). The electrophysiological criteria for diagnosing ARVC include epsilon waves, late potentials, prolonged terminal activation duration, ventricular tachycardia, and extrasystoles (Marcus et al., 2010). Diagnosis of ARVC depends on patients displaying a subset of these electrical abnormalities. Separate studies on heart biopsies from ARVC patients have shown that these defects can occur in the setting of ultrastructural defect in the ICD within the working myocardium; however, the status of cell–cell junctions has not been directly assessed in the CCS (e.g., His-Purkinje network) of ARVC hearts. Mutations in another desmosomal gene, plakophilin 2, have also been correlated with Brugada Syndrome, which is characterized by ST-segment abnormalities in leads V1–V3 on ECG analysis and ventricular arrhythmias (Cerrone et al.,

References

Cerrone et al. (2013) Nogami et al. (1990) Takemura et al. (2008) Balderramo and Caeiro (2004), Morady et al. (1984) Zusterzeel et al. (2013) Quarta et al. (2011)

Bae et al. (2013) Bao et al. (2013) Cox et al. (2011)

Kass et al. (1994) Makita et al. (2012)

2013), highlighting an impact of desmosomal defects on the His-Purkinke network in the human heart. The gap junction protein, Cx40, has also been associated with His-Purkinje dysfunction in humans. Longitudinal human genetic studies on a familial conduction system disease with arrhythmias and dilated cardiomyopathy have implicated connexin 40 in the disease etiology (Kass et al., 1994). A separate study on progressive familial heart block type I, which is characterized by conduction disturbances in the His-Purkinje system similarly implicated Cx40 in disease pathogenesis (Makita et al., 2012). CONCLUSIONS AND FUTURE DIRECTIONS It is now well established that cardiomyocytes of the specialized CCS harbor both mechanical junctions (anchoring junctions) alongside previously well-established electrical channels (e.g., gap junction and ion channels). Data from genetic mouse models and human studies have highlighted a potential functional role for anchoring junction components in the CCS; however, specific studies dissecting the role of these structures specifically within the CCS in the mouse and human are still lacking. Future studies focused on generating and characterizing CCS-specific mouse models targeting these components alongside electrophysiologically analyzing CCS function in patients harboring cardiac disease will provide much added insight into uncovering the mechanisms underlying human cardiac diseases associated with cell– cell junction defects.

CELL JUNCTIONS IN CARDIAC CONDUCTION SYSTEM Declaration of interest: J.P. was a previous recipient of the NHLBI-Graduate Research Assistant Diversity Supplement (NIH 3R01HL095780-01S2) and is currently funded by a NIH F31 Ruth L. Kirschstein National Research Service Award Graduate Fellowship (1F31HL120611-01). Funding for F.S. is provided by the National Institute of Health (NIH 1R01HL095780-01), California Institute of Regenerative Medicine (CIRM RB3-05103) and Saving tiny Heart Society grants.

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