Adherens Junctions

Adherens junctions are specialized structures essential for the mechanical coupling between neighboring cells. The three morphologically different forms of adherens junctions are puncta adherentia, zonula adherens, and fascia adherens, with the last name corresponding to the morphology found in the cardiac intercalated disc.²⁶ Cell-cell mechanical anchoring occurs at two crucial points: the extracellular space, within which cadherins tightly bind to each other, and the intracellular space, within which the cytoplasmic end of cadherin is indirectly attached to the actin cytoskeleton. The association between cadherin and the cytoskeleton involves at least two molecular “hinges”; cadherin binds to β-catenin and plakoglobin, and both molecules in turn bind to α-catenin (among others), the latter being in direct contact with actin. This is only a simplified description, because other interactions are likely to occur.²⁷ This string of intermolecular interactions provides mechanical continuity between cells, allowing for the mechanical work of individual myocytes to integrate into the pumping function of the heart.

Desmosomes

The desmosome (macula adherens) appears as two parallel tripartite plaques containing an intercellular gap of approximately 30 nm bisected by a distinct line, parallel to the apposed cell membranes (see Figure 22-1).²⁸ Desmosomes contribute to mechanical continuity between cardiac cells. Whereas adherens junctions link the actin cytoskeleton of adjacent cells, desmosomes provide continuity to the intermediate filament network (mainly desmin, in the case of heart).^28,29 In the extracellular space, desmosomal cadherins (desmocollins and desmogleins) bind tightly to each other. In the intracellular space, the intermediate filaments bind to desmoplakin. The interaction between desmoplakin and the desmosomal cadherin can be in some cases direct, but it mostly occurs through their association with plakophilin and plakoglobin.^28,29 The topologic organization of desmosomal molecules was studied by North et al.³⁰ using quantitative immunogold electron microscopy. More recently, Al-Amoudi et al.³¹ solved the three-dimensional molecular structure of the desmosomal plaque. Overall, structural and biochemical evidence combined show that desmoplakin binds to plakophilin through their N-terminal domains,^28,32 whereas desmoplakin binds to the intermediate filament by way of its C-terminal domain,^28,31 yielding a highly organized structure.

Hatsell and Cowin²⁹ once described the desmosome as “a system as staid and solid as the queen’s corsets.”²⁹ With that analogy, it is easy to imagine the loss of containment that would follow in its absence. Mice deficient in plakoglobin, desmoplakin, or plakophilin-2 (PKP2), die during embryonic development as a result of severe myocardial rupture.^33–35 More relevant from the point of view of clinical cardiology, ARVC in humans has been linked to mutations in desmosomal proteins.³⁶ The relation between various complexes of the intercalated disc and ARVC is discussed later in this chapter and in other review articles.^24,37

The Area Composita

Recent immunoelectron microscopy studies revealed the presence of a structure with mixed features of desmosomes and adherens junctions, dubbed the area composita.³⁸ This structure is found only in the heart of higher vertebrate species including mouse and humans.^39,40 The combination of components of the desmosomes and the adherens junctions allows anchorage of actin and desmin filaments to the same point, perhaps providing additional strength and flexibility to the muscle cell. Knockdown of PKP2 in neonatal cardiomyocytes leads to remodeling of the area composita.⁴¹ Additional studies suggest a role for α-catenin in the maintenance of these hybrid junctions.⁴² Interestingly, knockdown of α-catenin leads to PKP2 decrease only at the area composita and not at the desmosomes, suggesting that the molecular composition of the area composita, and its regulation, can be independent from that of other junctions.⁴² The area composita may represent a physical space where mechanical junction proteins interact with ion channel complexes.

Transcriptional Regulation by Mechanical Junction Proteins

Catenins are a part of both adherens junctions and desmosomes, thus participating in cell adhesion; however, these proteins also act as transcriptional activators. A prominent example is the participation of β-catenin in canonical Wnt signaling.⁴³ Binding of Wnt to its Frizzled receptor leads to an increase in levels of cytosolic β-catenin and a consequent translocation of the protein to the nucleus, where it binds to Tcf/Lef complex and promotes the expression of various genes such as c-Myc or c-Fos. In the heart, the Wnt signaling and the activation of genes by β-catenin/Tcf/Lef has been associated with the regulation of physiologic and pathologic growth of the cardiomyocytes.⁴⁴ Plakoglobin, another protein of the armadillo family, shows high homology with β-catenin and has been associated with the Wnt signaling. Different studies have shown that plakoglobin interacts and competes with β-catenin at multiple levels, acting as an antagonist of the Wnt/β-catenin signaling.^45–47 The fact that desmosomal proteins are involved in the regulation of the Tcf/Lef complex has been invoked as a possible mechanism for the fibrofatty infiltration common in hearts affected with ARVC.^48–50

Gap Junctions

In 1958, Sjostrand et al.⁴ described an area of specialization in the cardiac intercalated disc composed of “three dark lines with two intervening less dense lines”. This structure, which was similar to the one previously identified in the giant axon of the crayfish, was named the “longitudinal connexion” by these investigators. Years later, Revel coined the term gap junctions, thus emphasizing two key features: a gap between the cells and a junction between them.

Gap junctions form intercellular channels that provide a low-resistance pathway for direct cell-to-cell passage of electrical charge between cardiac myocytes. Each gap junction channel is composed of two hexameric structures called connexons that dock across the extracellular space and form a permeable pore isolated from the extracellular space. Each connexon results from oligomerization of an integral membrane protein, connexin. The most abundant connexin isotype in the heart, brain and other tissues is the 43-kD protein, connexin43 (Cx43).

The importance of Cx43 in the propagation of the cardiac action potential is well established. If Cx43 channels are not present, normal propagation is disrupted and lethal arrhythmias can ensue.51,52 Gap junction remodeling has been studied for various inherited and acquired diseases.^{10,11,14,53–55} The implications of connexin remodeling to electrophysiology are discussed later in this chapter.

Intercellular Space

The size of the space separating two cardiac cells at the intercalated disc changes depending on the proximity to the various structures, as well as the vesicular activity between the two cells (see Figure 22-1). It is a common view that the intercellular space is not relevant for electrophysiology. This view, however, is changing. Mathematical modeling studies^56,57 and experimental evidence⁵⁸ support the idea that the intercellular space is critical to propagation via an electric field mechanism.⁵⁹ This model will be discussed later. Of note, increased size of the intercellular space has been reported in animal models of ARVC.^20,21,60,61

Voltage-Gated Sodium Channel Complex

In 1996, Cohen⁶² showed that cardiac sodium channel proteins were preferentially localized at the intercalated disc,⁶² although they are also present over the cell surface, following a striated pattern.⁶³ Recent data emphasize how the VGSC interacts also with scaffolding, anchoring, and adhesions proteins, which regulate its function.

Potassium Channels at the Intercalated Disc

In 1995, Mays et al.⁷³ described for the first time that the potassium channel protein K_V1.5 localizes to the intercalated disc of adult myocardial cells. Later studies showed that K_V1.5 associates with SAP97.⁷⁴ Interestingly, SAP-97 also associates with Na_V1.5,⁷¹ making this protein a candidate for mutual regulation of both depolarization and repolarization channels, as is the case of Na_V1.5 and Kir2.1.⁷⁵ Additional studies have shown that the function of K_V1.5 depends on the expression of N-cadherin,⁷⁶ an interesting parallelism to the interaction between PKP2 (a desmosomal molecule), and Na_V1.5.⁷² Cheng et al.⁷⁶ also showed that cortactin is required for the N-cadherin–dependent regulation of K_V1.5. Of note, mice deficient in kcne2, an ancillary potassium channel subunit, display an impaired ventricular repolarization because of inhibition in the trafficking to the membrane of K_V1.5.⁷⁷

Another voltage-gated potassium channel that preferentially (though not exclusively) localizes to the intercalated disc is K_V4.2; it is responsible for the rapid repolarization phase of the cardiac action potential.78,79 Finally, regarding Kir channels, two different subunits localize at the transverse tubules and at the intercalated disc in canine myocytes: Kir2.1 and Kir2.3.⁸⁰

Desmosomal Proteins Are Necessary for the Formation of Functional Gap Junctions

Studies on the relation between desmosome integrity and cardiac electrophysiology were motivated by the finding that most familial cases of ARVC where a genetic link has been found, result from mutations in genes coding for desmosomal molecules. Dr. Saffitz and his colleagues were first to propose a link between mechanical and electrical junctions.54,81 In a series of seminal studies, these investigators first provided evidence that the molecular phenotype of the ARVC-afflicted heart involves not only desmosomes, but other molecules of the intercalated disc as well.^53,54 In particular, the authors showed a significant decrease in the abundance of Cx43 immunoreactive protein in the intercalated disc region of heart tissue obtained from affected patients. The hypothesis of a desmosome–gap junction crosstalk was confirmed in vitro by Oxford et al.,⁸² who used RNA silencing technology to reduce PKP2 expression in cardiac ventricular myocytes, as well as in epicardium-derived cells obtained from neonatal rat hearts. Their data showed that the loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separately, they demonstrated that Cx43 and PKP2 coexist in the same macromolecular complex. Follow up studies confirmed this observation,^41,83,84 giving support to the notion that two complexes previously considered independent are in fact, functionally and molecularly interactive. Recent studies on samples obtained from hearts affected with ARVC have confirmed the notion that in most (though not all) cases, there is a significant loss of Cx43 immunoreactive signal associated with the loss of desmosomal integrity in the cardiac intercalated disc.^85,86

Quantitative analysis in experimental models indicated that complete loss of PKP2 expression led to an approximate 50% decrease in gap junction–mediated cell-cell coupling.⁸² Previous studies had shown, however, that a 50% reduction in electrical coupling does not lead to significant changes in conduction velocity.^87–89 We therefore speculated that in addition to interacting with gap junctions, desmosomal molecules can also interact with ion channel complexes that reside at the intercalated disc.

Desmosomal Molecules Are Necessary for Sodium Channel Function

The observations described earlier led to the question of whether Cx43 is the only molecule relevant to electrophysiology that interacts with PKP2. Given the preferential localization of the VGSC complex at the intercalated disc, we explored its possible cross-talk with desmosomal molecules. Single, adult cardiac myocytes were treated with small interfering RNA (siRNA) to prevent expression of PKP2. Control cells were treated with a nonsilencing construct. As shown in Figure 22-2, A, the amplitude of I_Na was significantly reduced in cells lacking PKP2 expression. This reduction in amplitude was observed across the voltage range (see Figure 22-2, B). In addition, loss of PKP2 expression caused a negative shift in the voltage dependence of steady-state inactivation (see Figure 22-2, C), and a slowing of recovery from inactivation (see Figure 22-2, D). These changes in the major excitatory current were reflected in a decrease in the velocity of action potential propagation in monolayers of neonatal rat ventricular myocytes (Figure 22-3). Interestingly, computer simulations showed that the changes in amplitude and kinetics of the I_Na represented the key substrate for the generation of reentrant activity in a two-dimensional model of cardiac cells.⁹⁰ Overall, these data led us to propose that there is a functional crosstalk between a protein defined in the context of intercellular junctions (PKP2), and another complex primarily involved in supporting cell excitability (the VGSC complex). The results supported the hypothesis that arrhythmias that occur in patients with ARVC may have as a substrate not only changes in the macroscopic architecture of the tissue (as it would be expected once the fibrofatty infiltrations populate the heart), or in the integrity of intercellular coupling, but also changes in the electrical properties of the cardiac myocytes. Of relevance, these results revealed that a property “of the single cell” (excitability; inward sodium current [INa]) is in fact subject to modulation by proteins classically defined as belonging to the group of intercellular junction molecules.

PKP2 mutations associated with ARVC have all been found in only one allele. We therefore characterized the relation between PKP2 abundance and sodium current function in mice that were haploinsufficient for the pkp2 gene (PKP2-Hz).²⁰ Of note, one of the most common mutations in PKP2 is the presence of a stop codon at amino acid position 79 (R79x).⁹¹ This early truncation is functionally equivalent to haploinsufficiency.⁹² Patch clamp experiments showed a decreased amplitude and a shift in gating and kinetics of I_Na in PKP2-Hz myocytes, compared with control myocytes. To further unmask I_Na deficiency, we exposed myocytes, Langendorff-perfused hearts and anesthetized animals to a pharmacologic challenge (flecainide). In PKP2-Hz hearts, the extent of flecainide-induced I_Na block, impaired ventricular conduction, and altered electrocardiographic parameters were larger than controls. As shown in Figure 22-4 and described by Cerrone et al.,²⁰ flecainide provoked ventricular arrhythmias and death in PKP2-Hz animals, but not in wild type. These results showed that PKP2 haploinsufficiency leads to I_Na deficit in murine hearts, thus documenting for the first time the relation between the “desmosomal molecule” PKP2 and the VGSC complex in a living heart. Our results supported the contention that I_Na dysfunction contributes to generation and maintenance of arrhythmias in patients with desmosomal deficiency. It remains unclear whether pharmacologic challenges could help to unveil arrhythmia risk in patients with mutations or variants in PKP2.

The relation between desmosomal molecules and the VGSC has also been demonstrated in mice overexpressing a desmoglein-2 (DSG-2) mutation.²¹ This model is interesting given that severe arrhythmias and sudden death in these mice often occur before structural damage; the latter mimics a phenomenon often observed in humans affected with ARVC, where arrhythmias and electrocardiographic changes have been described early in the history of the disease, before overt structural changes in the myocardium.^24,54,93 Electrophysiological analysis of these DSG-2 transgenic mutant mice revealed prolonged ventricular activation time, decreased conduction velocity in both longitudinal and transverse directions, and increased arrhythmia susceptibility, prior to signs of fibrosis or necrosis in the myocardium. The authors also observed a decrease in maximal upstroke velocity and decreased I_Na amplitude. Taken together, the data demonstrate that a decrease in PKP2 abundance,²⁰ as well as the overexpression of a DSG-2 mutation,²¹ lead to impaired I_Na function in hearts with no histologic features of ARVC. These data provide a demonstration in vivo of the interaction between desmosomal molecules and the VGSC, and they suggest impaired sodium current as a substrate for lethal arrhythmias in the concealed phase of ARVC, as proposed earlier.⁷²

The question remains as to whether other desmosomal proteins also interact with Na_V1.5, and whether these interactions occur in the human heart. Recently, Gomes et al.⁹⁴ reported that mice that are haploinsufficient for desmoplakin present average peak I_Na density similar to control; however, careful analysis of their results suggests the possibility of technical limitations in their voltage clamp recordings, which could have masked small differences between the groups.²⁰ However, in the same study, the authors showed that patients with heterozygous mutations in desmoplakin and without overt structural disease had significant regional conduction delays and heterogeneous Na_V1.5 distribution.⁹⁴ The possibility of changes in the abundance or colocalization of Na_V1.5 in the intercalated disc area of the hearts of patients with ARVC is a matter of current investigation.

Connexin43 Regulates Sodium and Potassium Currents

Loss of Cx43 expression leads to propagation block and to arrhythmias. Interpretation of this result has centered mostly on the role of Cx43 as the pore-forming subunit of gap junctions. However, the latter does not exclude the possibility that Cx43 could also interact with other channel complexes, and affect their function. In fact, there is no reason to confine Cx43 to a single task, in a single structure.

The first indication of an interaction between Cx43 and the VGSC came from the work of Malhotra et al., showing coprecipitation of Na_V1.5 with Cx43.⁹⁵ The physical proximity of these molecules was recently confirmed by Rhett et al.⁹⁶ Not only are Cx43 and Na_V1.5 in close proximity, but these two molecules are also functionally intertwined. Indeed, as shown in Figure 22-5, Jansen et al.¹⁷ recently reported that siRNA-mediated loss of Cx43 expression in adult ventricular myocytes leads to a decrease in the amplitude of the I_Na. The functional effect coincided with decreased colocalization of Cx43 and Na_V1.5 at the intercalated disc. A similar decrease in I_Na was later reported by Desplantez et al.¹⁸ in fetal atrial myocytes of Cx43-deficient mice.¹⁸ Overall, the data demonstrate that Cx43 expression is necessary for proper sodium current function, and for the accumulation of Na_V1.5 at the cardiac intercalated disc. Interestingly, the regulation of I_Na by Cx43 is reciprocated by the fact that ankyrin-G, a molecule that is well characterized as a component of the VGSC complex, is necessary to preserve gap junction–mediated coupling between neonatal myocytes.⁶⁷

The VGSC is not the only electrically functional complex of the intercalated disc that is disrupted consequent to loss of Cx43 expression. In fact, the first report correlating Cx43 expression to nonjunctional currents was by Danik et al.¹⁶ These authors noted that the action potential duration recorded from the ventricle of Cx43 conditional knockout animals were significantly shorter than control. This shortening associated with higher levels of sustained repolarizing current and higher levels of inward rectifier current in myocytes from the right ventricle. Overall, the data show that Cx43 is not only a gap junction–forming molecule in the heart but also, a component of a molecular network that regulates excitability and repolarization.

AnkG and Cx43 Are Necessary to Maintain Intercellular Adhesion

The previous sections have described that molecules of the desmosome, relevant to intercellular adhesion (namely PKP2 and DSG-2), are actually important to preserve I_Na amplitude and electrical coupling between cardiac cells. We then speculate that, if the system works as a unit, decreased protein levels of either Cx43 or a component of the VGSC would affect intercellular adhesion strength. Consistent with this hypothesis, we have shown that intercellular adhesion strength is decreased in monolayers of neonatal rat ventricular myocytes treated with siRNA to prevent expression of AnkG.⁶⁷ Similarly, loss of Cx43 expression in cultured cells significantly impaired intercellular adhesion strength,⁷ a result consistent with previous observations.⁹⁷

The “α Personality” of the β-Subunit: Intercellular Adhesion and the Sodium Current

The finding that mutations in desmosomal molecules associate with familial cases of ARVC has highlighted the important link between cell adhesion and sodium channel function. In retrospect, the link between these two seemingly unrelated functions was first established several years ago by the Isom lab. In 1981, Hartshorne and Caterall⁹⁸ purified “the saxitoxin receptor of the sodium channel from rat brain” and identified two polypeptides, which they referred to as “α” and “β.”⁹⁸ They proposed that these two subunits conformed the functional sodium channel. In 1992, Isom et al.⁹⁹ isolated the cDNA, sequenced and functionally expressed the β-1 subunit, concluding that this protein is crucial to the overall function of the sodium channel. In this manner, this 22,581-d protein was labeled as a “β” for its “α,” a subunit merely accessory to sodium channel function. It was 8 years later (in 2000) that the Isom lab demonstrated that “sodium channel beta subunits” also mediate cell adhesion,¹⁰⁰ an important fact in the formation of the sodium channel complex^100,101 and in sodium channel–independent functions such as cell migration, cell aggregation, and interaction with the cytoskeleton.¹⁰² Studies that followed demonstrated their critical role in cancer.¹⁰³ Interestingly, the fact that β-subunits are actually independent adhesion molecules has, for the most part, missed the attention of cardiac electrophysiologists. Another case of a molecule’s identity being boxed into the function related to its original discovery. If that molecule had been found through cell adhesion assays and called adherin, perhaps its role as a modulator of I_Na could have gone unnoticed for years. The fact is that the sodium channel β-subunits are, together with PKP2, DSG-2, AnkG, and Cx43, members of a growing family of molecules that regulate sodium channel function and intercellular adhesion strength. Whether β-subunits regulate gap junction–mediated coupling (as the other molecules listed earlier) is a matter of future investigation.

Other Intercellular Adhesion Molecules That Cross-Talk with Cardiac Ion Channels

Two other adhesion molecules have been associated with cardiac electrical function: N-cadherin and the coxsackievirus and adenovirus receptor (CAR). N-Cadherin is well recognized as critical to the mechanical coupling between cells. However, Li et al.¹⁰⁴ showed that restricted cardiac deletion of N-cadherin also leads to severe arrhythmias, and a loss of Cx43 from the intercalated disc. More recently, the Radice lab reported that in addition to effects on coupling, loss of N-cadherin leads to decreased density of the repolarizing current I_K.slow, concurrent with decreased expression of K_V1.5 and its accessory protein Kcne2.⁷⁶ The properties of the I_Na in cadherin-deficient hearts remain undefined.

CAR is another case of a molecule whose identity is burdened by its birth name. Undoubtedly a receptor for both coxsackievirus and adenovirus, studies have demonstrated that the immunoglobulin extracellular domains of CAR are capable of homophilic binding and participate in intercellular adhesion in epithelial cells. The role of this molecule on cell adhesion in the heart is less defined. Interestingly, cardiac-restricted deletion of CAR causes significant slowing of A-V propagation and disruption of gap junctions at the intercalated disc.105,106 Research is in progress to determine whether, as other molecules with immunoglobulin extracellular domains (such as the sodium channel β-subunits), CAR expression modulates sodium current function.

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