Key Words: plakophilin-2 Ⅲ intercalated disc Ⅲ arrhythmogenic right ventricular cardiomyopathy Ⅲ cardiac desmosomes A high-resolution image of the site of end-end contact between cardiomyocytes reveals an electron-dense organization called "the intercalated disc." Its classic definition involves 3 structures: desmosomes and adherens junctions, providing mechanical coupling; and gap junctions, allowing electric/metabolic synchronization between cells. Recent studies show that other molecules, not directly involved in intercellular coupling, also reside preferentially at the intercalated disc. Among them is Na V 1.5, the major ␣ subunit of the cardiac sodium channel. 1 Here, we ask whether Na v 1.5 and the desmosomal protein plakophilin-2 (PKP2) coexist in the same molecular complex and whether loss of PKP2 expression affects (1) the amplitude and kinetics of the sodium current and (2) action potential propagation in a monolayer of cardiomyocytes. Our data demonstrate a functional crosstalk between a protein defined in the context of intercellular junctions (PKP2) and another protein that is fundamental to the electrical behavior of the single myocyte.

In pathological conditions such as ischemic cardiomyopathy and heart failure, differentiation of fibroblasts into myofibroblasts may result in myocyte-fibroblast electrical coupling via gap junctions. We hypothesized that myofibroblast proliferation and increased heterocellular coupling significantly alter two-dimensional cardiac wave propagation and reentry dynamics. Co-cultures of myocytes and myofibroblasts from neonatal rat ventricles were optically mapped using a voltage-sensitive dye during pacing and sustained reentry. The myofibroblast/myocyte ratio was changed systematically, and junctional coupling of the myofibroblasts was reduced or increased using silencing RNAi or adenoviral overexpression of Cx43, respectively. Numerical simulations in two-dimensional models were used to quantify the effects of heterocellular coupling on conduction velocity (CV) and reentry dynamics. In both simulations and experiments, reentry frequency and CV diminished with larger myofibroblast/myocyte area ratios; complexity of propagation increased, resulting in wave fractionation and reentry multiplication. The relationship between CV and coupling was biphasic: an initial decrease in CV was followed by an increase as heterocellular coupling increased. Low heterocellular coupling resulted in fragmented and wavy wavefronts; at high coupling wavefronts became smoother. Heterocellular coupling alters conduction velocity, reentry stability, and complexity of wave propagation. The results provide novel insight into the mechanisms whereby electrical myocyte-myofibroblast interactions modify wave propagation and the propensity to reentrant arrhythmias.

pH-induced closure of connexin43 (Cx43) channels involves interaction of the Cx43 carboxyl-terminal (Cx43CT) with a separate "receptor" domain. The receptor location and structure and whether the interaction is directly intramolecular are unknown. Here we show resonant mirror technology, enzyme-linked sorbent assays, and nuclear magnetic resonance (NMR) experiments demonstrating pH-dependent binding of Cx43CT to region 119 -144 of Cx43 (Cx43L2), which we propose is the receptor. NMR showed that acidification induced ␣-helical order in Cx43L2, whereas only a minor modification in Cx43CT structure was detected. These data provide the first demonstration of chemically induced structural order and binding between cytoplasmic connexin domains.

Abstract-Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts.We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that 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. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins is an inherited disease that presents with sustained monomorphic ventricular tachycardia and sudden cardiac death. The disease is characterized by progressive fibrofatty infiltration of the myocardium, most prominent in the free wall of the right ventricle. 1 Recent studies have linked ARVC with mutations in proteins of the cardiac desmosome, 2 a component of the intercalated disc essential for mechanical coupling between cardiac cells. 3 It is estimated that as many as 70% of the mutations linked to familial ARVC are in the gene coding for plakophilin (PKP)2, 4 a 98-kDa desmosomal protein. PKP2 interacts with plakoglobin, desmoplakin, and the desmosomal cadherins via its amino terminal ("head") domain. [5][6] Loss of PKP2 destabilizes the desmosome, 7 and its genetic deletion in mice leads to rupture of the myocardial wall during the embryonic stage. 7 Loss of desmosomal integrity could lead to disruption of mechanical function in hearts afflicted with ARVC; yet, the latter does not directly explain the highly arrhythmogenic nature of the disease, particularly in cases in which lifethreatening arrhythmias occur in the absence of severe displacement of myocardium with fatty or fibrous tissue. 8 Recently, Saffitz and colleagues proposed that disruption of mechanical coupling may lead to loss of gap junctionmediated electrical communication between cells. 8 -10 This hypothesis awaits confirmation in a cellular model in which protein expression can be manipulated and intercellular communication can be assessed directly.Here, we used small interfering (si)RNA technology to silence PKP2 expression in neonatal cardiac cells, and we explored the effect of loss of PKP2 expression on the distribution and function of gap junctions. Our studies focused primarily on 2 cell populations: cardiac myocytes and epicardium-derived cells (EPDCs). Although the importance of cardiac myocytes in the context of ARVC and arrhythmias seems self-evident, a possible role for EPDCs in ARVC has not been described. Yet, as progenitors of the cardiac fibroblast cell lineage, the function of EPDCs deserves atte...

Rationale The early description of the intercalated disc defined three structures, all of them involved in cell-cell communication: desmosomes, gap junctions and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells, also populate the intercalated disc. Key among them is the voltage-gated sodium channel (VGSC) complex. An important component of this complex is the cytoskeletal adaptor protein ankyrin-G (AnkG). Objective To test the hypothesis that AnkG partners with desmosome and gap junction molecules, and exerts a functional effect on intercellular communication in the heart. Methods and Results We utilized a combination of microscopy, immunochemistry, patch clamp and optical mapping to assess the interactions between AnkG, plakophilin-2 (PKP2) and Connexin43 (Cx43). Co-immunoprecipitation studies from rat heart lysate demonstrated associations between the three molecules. Using siRNA technology we demonstrated that loss of AnkG expression caused significant changes in subcellular distribution and/or abundance of PKP2 and Cx43, as well as a decrease in intercellular adhesion strength and electrical coupling. Regulation of AnkG and of Nav1.5 by PKP2 was also demonstrated. Finally, optical mapping experiments in AnkG-silenced cells demonstrated a shift in the minimal frequency at which rate-dependence activation block was observed. Conclusions These experiments support the hypothesis that AnkG is a key functional component of the intercalated disc, at the intersection of three complexes often considered independent: the VGSC, gap junctions and the cardiac desmosome. Possible implications to the pathophysiology of inherited arrhythmias (such as arrhythmogenic right ventricular cardiomyopathy; ARVC) are discussed.

Abstract-The inwardly rectifying potassium (Kir) 2.x channels mediate the cardiac inward rectifier potassium current (I K1 ). In addition to differences in current density, atrial and ventricular I K1 have differences in outward current profiles and in extracellular potassium ([K ϩ ] o ) dependence. The whole-cell patch-clamp technique was used to study these properties in heterologously expressed Kir2.x channels and atrial and ventricular I K1 in guinea pig and sheep hearts. Kir2.x channels showed distinct rectification profiles: Kir2.1 and Kir2.2 rectified completely at potentials more depolarized than Ϫ30 mV (IϷ0 pA). In contrast, rectification was incomplete for Kir2.3 channels. In guinea pig atria, which expressed mainly Kir2.1, I K1 rectified completely. In sheep atria, which predominantly expressed Kir2.3 channels, I K1 did not rectify completely. Single-channel analysis of sheep Kir2.3 channels showed a mean unitary conductance of 13.1Ϯ0.1 pS in 15 cells, which corresponded with I K1 in sheep atria (9.9Ϯ0.1 pS in 32 cells Key Words: Kir2 Ⅲ extracellular potassium Ⅲ heteromerization Ⅲ rectification I n the heart, the inwardly rectifying potassium (Kir) current (I K1 ) stabilizes the resting membrane potential and plays a major role during the final phase of action potential (AP) repolarization. 1-3 The Kir2.x channels mediate cardiac I K1 . 3 Previous studies have demonstrated that I K1 properties are different in atrial and ventricular myocytes. 1,[3][4][5][6] First, I K1 current density is higher in the ventricles than in the atria. 6,7 Second, ventricular I K1 has been described as having a more prominent negative slope conductance at depolarized potentials than atrial I K1 (ie, atrial I K1 does not rectify completely). 1,4,5 Also, the outward component of the background potassium current (I B; consisting mainly of I K1 ) is significantly increased in high extracellular potassium ([K ϩ ] o ) in ventricular but not atrial myocytes. 1 The molecular mechanisms underlying these I K1 differences are unknown.The Kir2.x channel expression patterns may determine outward I K1 properties. 8,9 Outward currents through Kir channels may play an important role in the dynamics of atrial and ventricular fibrillation, as studied in the sheep 10 and guinea pig, 11 respectively. However, outward current profiles of the individual Kir2.x isoforms have not been comparatively studied. Moreover, the effect of high [K ϩ ] o on outward currents of Kir2.x isoforms has also not been compared. It is possible that the properties of Kir2.x isoforms, existing either as homomers or heteromers, determine regional I K1 differences in the heart. Although recent studies have shown that Kir2.x subunits heteromerize, 12- .3 were cloned using the polymerase chain reaction and transiently transfected into human embryonic kidney 293 (HEK293) cells using the Qiagen Effectene protocol. Guinea pig and sheep cardiac myocytes were enzymatically dissociated using the Langendorff-retrograde perfusion method as described previously. 11 In...

Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric͞homotypic channels with distinct permeability and gating properties but do not form functional homomeric͞heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric͞homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.Gap junction channels connect the cytoplasms of adjacent cells and provide a pathway for intercellular diffusion of ions, second messenger molecules, and small metabolites. Functional gap junction channels are formed by connexins (Cxs), a gene family with at least 14 mammalian members that are distinguished from one another by their predicted molecular weights expressed in kilodaltons (e.g., Cx43, the 43-kDa connexin). Cxs oligomerize to form connexons (hemichannels), which are defined as homomeric when the six comprising Cxs are identical or heteromeric when two or more Cxs comprise the connexon. Connexons in adjacent cells join in the extracellular space to form the functional intercellular channel, which is defined as homotypic when the Cx composition of the contributing connexons is identical or heterotypic when different.The ability of Cxs to form homomeric͞heterotypic channels has been examined in the Xenopus oocyte and HeLa cell expression systems as well as in other settings (for review, see ref. 1). Homomeric Cx43 connexons successfully dock with homomeric connexons comprised of Cxs 30.3, 37, and 45 but not with Cxs 50, 40,33,32, 31.3,31,or 26. Homomeric Cx40 connexons successfully dock with and form functional homomeric͞heterotypic channels with Cxs 37 and 45 but not with Cxs 50, 46, 43,32, 31.1,31,or 26. Clearly, there are far more incompatible than compatible combinations.The capacity of Cxs to form functional heteromeric connexons and channels has only recently received attention. Biochemical and structural tools have demonstrated the existence of heteromeric connexons comprised of Cxs 46 and 50 (2) and Cxs 32 and 26 (3). That these Cx pairs form functional hetero...

Previous studies have shown that chemical regulation of connexin43 (Cx43) depends on the presence of the carboxyl terminal (CT) domain. A particle-receptor (or "ball-and-chain") model has been proposed to explain the mechanism of gating. We tested whether the CT region behaved as a functional domain for other members of the connexin family. The pH sensitivity of wild-type and Ct-truncated connexins was quantified by use of electrophysiological and optical techniques and the Xenopus oocyte system. The CT domain of Cx45 had no role in pH regulation, although a partial role was shown for Cx37 and Cx50. A prominent effect was observed for Cx40 and Cx43. In addition, we found that the CT domain of Cx40 that was expressed as a separate fragment rescued the pH sensitivity of the truncated Cx40 (Cx40tr), which was in agreement with a particle-receptor model. Because Cx40 and Cx43 often colocalize and possibly heteromerize, we tested the pH sensitivity of Cx40tr when coexpressed with the CT domain of Cx43 (hetero-domain interactions). We found that the CT domain of Cx43 enhanced the pH sensitivity of Cx40tr; similarly, the CT domain of Cx40 restored the pH sensitivity of the truncated Cx43. In addition, the CT domain of Cx43 granted insulin sensitivity to the otherwise insulin-insensitive Cx26 or Cx32 channels. These data show that the particle-receptor model is preserved in Cx40 and the regulatory domain of one connexin can specifically interact with a channel formed by another connexin. Hetero-domain interactions could be critical for the regulation of heteromeric channels.