Cell Tissue Res DOI 10.1007/s00441-014-2020-8

REVIEW

Connexins in the heart Pier D. Lambiase & Andrew Tinker

Received: 28 August 2014 / Accepted: 24 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Connexins are essential in the propagation of electrical activity throughout the heart and are an important determinant of conduction velocity. Their dysfunction is an important factor in the genesis of abnormal cardiac rhythm and is relevant to the pathogenesis of a wide variety of cardiac pathologies. Here, we review the basic biology of connexins in the heart but particularly focus on their abnormal function in cardiac disease.

Keywords Heart . Connexins . Conduction . Arrhythmia . Intercalated disc

Abbreviations ARVC Arrhythmogenic right ventricular cardiomyothy AF Atrial fibrillation Cx43 Connexin43 NOS1AP Nitric oxide synthase-1 adaptor protein VF Ventricular fibrillation VT Ventricular tachycardia

The work in the authors’ laboratories is or has been supported by the British Heart Foundation, Medical Research Council, Wellcome Trust, The National Institute for Health Research Barts Cardiovascular Biomedical Research Unit, UCLH Biomedicine NIHR and Heart Research UK. P. D. Lambiase Institute of Cardiovascular Science, Heart Hospital, UCL, 16-18 Westmoreland Street, London W1G 8PH, UK e-mail: [email protected] A. Tinker (*) William Harvey Heart Centre, Barts & The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK e-mail: [email protected]

Introduction Gap junctions are membrane pores providing electrical continuity between two cells: they are non-selective, typically allowing molecules of less than 1000 Daltons to transit, and have high ionic conductance. They are made up of a family of proteins called connexins and are classified according to their molecular weight. Over twenty isoforms have been recognised but four contribute to the heart, namely Cx43, Cx40, Cx45 and Cx30.2 (Davis et al. 1994; Jansen et al. 2010). The pore of the channel in each cell membrane is a hexamer with each cell contributing one hemichannel hexameric unit to the mature gap junction. The individual monomer is an integral membrane protein with four transmembrane domains and intracellular N- and C-termini. In cardiac myocytes, connexins are preferentially located at the intercalated disc in plaque-like arrangements (Rhett et al. 2013). They account for the anisotropic nature of conduction whereby conduction in the longitudinal direction of myocyte alignment is faster than that in the transverse (Saffitz et al. 1995; King et al. 2013). In this review, we largely focus on Cx43 and ventricular arrhythmia.

Distribution and function in cardiac cells Cx43 is the major isoform in the heart and is present in ventricular and atrial myocardium, whereas Cx40 is expressed in atrial myocytes, the atrioventricular node and His-Purkinje system (Beyer et al. 1987; Gourdie et al. 1991; Davis et al. 1994). Cx45 is present selectively in pacemaker and conducting tissues, namely in the sinoatrial node, atrioventricular node and His-Purkinje system (Coppen et al. 1999). Cx30.2 is present in the murine sinoatrial node but this may well be unique to rodents (Kreuzberg et al. 2005, 2009). In the discussion of function, we initially and largely

Cell Tissue Res

focus on Cx43 as this is very much the paradigm and has been the best studied. Mice with global deletion of Cx43 die at birth because swelling and obstruction of the right ventricular outflow tract impairs pulmonary perfusion (Reaume et al. 1995). The study of mice with haploinsufficiency of Cx43 has led to conflicting results about the effects on conduction velocity with some investigators reporting slowing whereas others see no effect (Guerrero et al. 1997; Morley et al. 2000). In an effort to clarify the situation, a number of murine models have been developed with conditional deletion of Cx43 in the post-natal heart (Gutstein et al. 2001; Eckardt et al. 2004; Danik et al. 2004; van Rijen et al. 2004). The conclusion from these studies is that conduction velocity slowing and ventricular arrhythmias are not observed until substantial depletion of Cx43 has occurred. For example, no change in conduction velocity or arrhythmia inducibility is found when Cx43 levels have declined to ~60 % of control values but, at levels of ~20 %, conduction velocity is reduced by half and inducible ventricular tachycardia (VT) becomes apparent in 80 % of mice (Danik et al. 2004). Thus, considerable reserve is present in Cx43 expression before conduction is affected. No evidence of structural heart disease exists suggesting a purely electrical pathogenesis; this is contributed to purely by changes in conduction velocity as the effective refractory period does not change (Danik et al. 2004). However. a subsequent report has shown changes in action potential duration and K+ currents in mice with conditional deletion of Cx43 suggesting that the electrical phenotype is more complex (Danik et al. 2008). Controversy remains as to whether Cx43 hemichannels are functional under physiological conditions and can release biologically active molecules such as ATP (Scemes 2012). As described above, although Cx43 is present in atrial myocytes, Cx40 seems to make a significant contribution to atrial conduction and abnormalities in its level and distribution may predispose to supraventricular arrhythmia. Furthermore, mice with genetic deletion of Cx40 consistently show delays in atrioventricular conduction. Cx45 seems only to make a contribution on a background of Cx40 deletion. Finally, in the sinoatrial node, redundancy seems to be present and it is difficult to pinpoint a specific connexin as being important. Substantial primary literature is available in these areas (also see below) and this is comprehensively summarised and discussed in a recent review (Jansen et al. 2010). The heart contains significant numbers of fibroblasts and, under pathological conditions, these cells may couple to cardiomyocytes. In an interesting study, fibroblasts were differentiated into myofibroblasts resembling those that can occur in vivo in pathological conditions such as hypertensive heart disease. These were then combined with purified cardiomyocytes and conduction velocity was measured in engineered fibres. The myofibroblasts expressed Cx43 and Cx45 and affected conduction velocity in a biphasic fashion

with a peak at low ratios of myofibroblast to cardiomyocytes (Miragoli et al. 2006). However, at high ratios, the changes were proarrhythmic with slowed conduction, re-entry and ectopic activity (Miragoli et al. 2007; Xie et al. 2009). Furthermore, myofibroblasts promoted fibrosis and this led to proarrhythmic local conduction delay and circuitous conduction pathways. Gap junctions are recognised in the ventricle as hexagonal semi-crystalline arrays of a number of Cx43 proteins present at the intercalated disc. In addition to connexins, the intercalated disc also contains desmosomes and fascia adherens, which maintain mechanical stability and cell-cell links between the cytoskeleton of one myocyte and the adjacent myocyte (Delmar and McKenna 2010). Furthermore, ion channels, particularly sodium channels (SCN5A, Nav1.5), which are also critical for impulse conduction, can concentrate at the intercalated disc (Malhotra et al. 2004). Evidence is emerging that non-channel proteins enriched in the intercalated disc influence the QT interval (Kapoor et al. 2014). What are the specific features of their interaction? The C-terminus of Cx43 interacts with the scaffolding protein called zonula occludens-1 around the gap junction array and might be involved in the stabilising of the formation of full gap junctions from hemichannels (Hunter et al. 2005). SCN5A also interacts with Cx43 but not via zonula occludens-1 (Malhotra et al. 2004). However, SCN5A and Cx43 are known to interact with both scaffolding proteins SAP97 and ankyrin G in cardiac myocytes (Sato et al. 2009, 2011; Asimaki et al. 2014). Arrhythmogenic right ventricular cardiomyothy (ARVC) is a disorder characterised by ventricular arrhythmias and sudden cardiac death. In the early phase of the disease, minimal structural abnormalities are apparent, although minor electrocardiographic changes are detectable (e.g. T wave inversion, epsilon waves, late potentials). As the disease progresses, patients develop regional wall motion abnormalities attributable to fibro-fatty replacement of the myocardium and overt ventricular arrhythmias. In recent years, ARVC has been shown to arise from mutations in structural proteins making up the desmosome. The integrity of the desmosome also seems able to influence the distribution of important functional molecules. In a murine model of desmin-related cardiomyopathy (resulting from a 7-amino-acid deletion of desmin) confocal microscopy demonstrated reductions in Cx43 at intercalated discs (Gard et al. 2005). The total tissue content of Cx43 and mechanical junction proteins were not reduced as quantified by immunoblotting, suggesting that diminished connexin expression at the intercalated discs was caused by a failure of these proteins to assemble or traffic properly. Optical mapping of the ventricle demonstrated that conduction was slowed uniformly in both transverse and longitudinal directions indicating that failed gap junction protein localisation had measurable negative effects upon conduction.

Cell Tissue Res

Significant prolongation of P wave duration and a trend towards QRS prolongation were seen, but no arrhythmias occurred on telemetry. A recent study of the heterozygous plakoglobin knockout mouse also demonstrated delayed conduction and increased ventricular arrhythmia (Kirchhof et al. 2006). These studies show that disruption of the desmosomal protein architecture results in abnormal gap junction assembly and functional conduction abnormalities that could provide the mechanistic basis for arrhythmia and sudden death in ARVC. Structural discontinuities, which are recognised in ARVC, have been shown to cause activation delay after premature stimulation, increasing susceptibility to wave break and ventricular fibrillation (VF; de Bakker et al. 1993; Derksen et al. 2003). Work from Delmar’s group demonstrates possible close associations between gap junction proteins and Na channels at the intercalated disc. The perinexus region adjacent to the intercalated disc is a newly described microdomain surrounding the cardiac gap junction and contains elevated levels of Cx43 and the sodium channel protein, Nav1.5 (Rhett et al. 2011). Biochemical analysis has demonstrated that plakophilin 2 co-immunoprecipitates not only with Cx43 but also with the major alpha-subunit of the cardiac sodium channel, Nav1.5 (Sato et al. 2009). Voltage clamp experiments have revealed that the loss of plakophilin 2 expression also leads to a decrease in amplitude and a shift in the voltage-gating kinetics of the sodium current in adult cardiac myocytes. Thus, Cx43 and SCN5A fail to traffic as efficiently to the intercalated disc in a number of models of ARVC (Gomes et al. 2012; Jansen et al. 2012; Cerrone et al. 2012). In a recent study, reduction in the inward rectifier current IK1 and mislocalisation of SAP97 were observed in a zebrafish model with a plakoglobin mutant. Kir2.1 is known to bind to SAP97 and this together with Cx43, SCN5A and plakoglobin localisation and electrophysiological properties are corrected by SB216763 (Asimaki et al. 2014). In another interesting recent study, nitric oxide synthase-1 adaptor protein (NOS1AP) was located at the intercalated disc and shown to influence conduction velocity (Kapoor et al. 2014). This protein has been implicated in the polygenic inheritance of QT interval as a trait and this study isolated an enhancer element that affected the expression of NOS1AP (Arking et al. 2006). Furthermore, in genetic analyses, a number of other intercalated disc proteins have been shown to be associated with QT interval determination (Kapoor et al. 2014). This suggests a link between the molecular components influencing cardiac repolarisation and the intercalated disc and conduction. These relationships are not totally interdependent, however, as it is possible conditionally to delete Cx43 and gap junctions in murine ventricle without affecting the formation of desmosomes and adherens junctions (Gutstein et al. 2003). Pathological studies in patients are reviewed in the section below. All of these data emphasise the need to develop a comprehensive model of the structural and functional interactions of

intercalated disc proteins. We think that this is an interesting area for exploration over the next few years.

Modulation of function Cx43 as a protein is rapidly turned over generally with a half-life of 1.3 h in cardiac myocytes (Beardslee et al. 1998). The anterograde transport of Cx43 is dependent on the direct delivery of Cx43 to the intercalated disc from the microtubule network (Shaw et al. 2007). Furthermore, these vesicles are stalled on the nonsarcomeric actin cytoskeleton and this interaction is crucial for forward trafficking and membrane delivery (Smyth et al. 2012). These processes are possibly disturbed in hearts subjected to ischaemia and oxidative stress (Smyth et al. 2010). Finally, internal translation of short isoforms, in particular a 20-kDa isoform might act as a chaperone in endoplasmic reticulum maturation (Smyth and Shaw 2013). In addition, Cx43 can redistribute from the intercalated disc to the sarcolemma (“lateralisation”) after delivery to the intercalated disc and is thought to be functionally inactive at this site (Beardslee et al. 2000). The phosphorylation of Cx43 through a number of kinase systems is considered to underlie these key events (Musil et al. 1990; MarquezRosado et al. 2012). Cx43 is predominantly phosphorylated on serine residues in the C-terminus of the protein (Lampe et al. 2006; Marquez-Rosado et al. 2012). The phosphorylation results in a shift in gel mobility and several different molecular species are apparent. The use of phosphorylation-specific antibodies has also demonstrated the importance of differential phosphorylation in the life cycle of the protein. Thus, phosphorylation is necessary for the assembly of functional connexins (Musil et al. 1990). Protein kinase C has been implicated as being critical and the phosphorylation of S368 impairs junctional communication (Lampe et al. 2000). Despite this, analogous phosphorylation mediated by protein kinase C also occurs in preconditioning, although at S262, and this might be important for some aspects of cellular protection (Srisakuldee et al. 2009). Extracellular signal-related kinase 1/2 and p38 mitogenactivated protein kinase might also contribute to these phenomena (Naitoh et al. 2009). Tyrosine phosphorylation at specific residues has further been shown to be responsible for the src-mediated downregulation of gap junction communication (Lin et al. 2001). Finally, the dephosphorylation of Cx43 might particularly contribute to changes seen in cardiac pathologies such as ischaemia and hypertension (see below) and this is possibly mediated by protein phosphatase 1 (Beardslee et al. 2000; Jeyaraman et al. 2003).

Cell Tissue Res

Mechanism of arrhythmogenesis The electrophysiological mechanisms leading to lethal ventricular arrhythmias and the factors accounting for sudden cardiac death in situations of impaired gap junction coupling remain uncertain. Cellular electrical uncoupling might unmask ectopic foci or trigger arrhythmias by enhancing the generation of early afterdepolarisations. Computer modelling indicates that moderate decreases in junctional coupling predispose to the generation of early afterdepolarisations, whereas higher levels of junctional resistance limit their propagation (Saiz et al. 1999). In vitro studies of dissociated myocytes have led to the hypothesis that extreme conduction slowing in combination with geometrical factors permits re-entrant excitation to take place in extremely small areas of cardiac tissue (Rohr et al. 1998). Two principal mechanisms of arrhythmia occur in diseased myocardium, namely re-entry and increased triggered activity attributable to calcium overload. Both gap junction lateralisation and local fibrosis are known to create ideal conditions for re-entrant tachycardias promoting slow conduction and unidirectional block. Studies of the healing canine epicardial infarct border zone have shown gap junction lateralisation and non-uniform transverse propagation (Smith et al. 1991; Peters et al. 1997). This leads to heterogeneous refractoriness in the myocardium and hence to conduction block and thus easily induced re-entrant arrhythmias. Furthermore, reduced cell coupling predisposes to enhanced dispersion of action potential duration and repolarisation, which creates the optimal conditions for re-entry (Viswanathan et al. 1999; Viswanathan and Rudy 2000). During ischaemia, gap junction uncoupling is recognised to occur after 15 min to be associated with (1) dephosphorylation of Cx43, (2) impulse propagation slowing, (3) increased anisotropy, (4) unidirectional block, (5) re-entrant arrhythmias and (6) VF (Smith et al. 1995; Marquez-Rosado et al. 2012). Intracellular resistance can triple and longitudinal conduction velocity can slow 2.5-fold. This creates large gradients of conduction and repolarisation dynamics that are highly pro-arrhythmic.

Drugs targeting gap junction function to prevent arrhythmias Optimisation of cellular electrical coupling would be predicted to ensure more homogeneous propagation of activation wavefronts and to minimise opportunities for re-entrant arrhythmias to develop. This is particularly pertinent to ischaemic myocardium where sudden death from malignant ventricular arrhythmias accounts for 300,000 deaths per annum in the USA alone, despite the advent of primary angioplasty to minimise infarct damage. The advent of such early interventions for coronary disease and myocardial infarction has created a population of patients who have localised regions of

myocardial damage and preserved left ventricular function but who are still at risk of sudden cardiac death. Class I and class III anti-arrhythmics have failed to prevent lethal arrhythmia and following CAST, class I agents are contraindicated in acute ischaemia (The Cardiac Arrhythmia Suppression Trial (CAST) Investigators 1989, 1992). Furthermore, apart from amiodarone, these agents cannot be employed in patients with impaired left ventricular function as they are negatively inotropic and associated with poor prognosis. This has spurred interest in developing alternative anti-arrhythmic agents. Gap junctions would provide a potentially suitable target, as upregulating gap junction expression/opening might optimise cellular electrical coupling and action potential propagation minimising the effects of the gap junction lateralisation and increased anisotropy seen in diseased tissue. Some evidence that gap junction modulation represents a therapeutic target has arisen from post-infarct trials of betablockers demonstrating reductions in mortality partly attributable to reduced sudden death events. Metoprolol increases Cx43 protein levels but not Cx43 mRNA, phosphorylation or kinase activation, suggesting that it stabilises Cx43 at the intercalated discs or prevents its degradation (Salameh et al. 2009). A number of anti-arrhythmic peptides have been identified or synthesised to modulate gap junction function. Aonuma et al. (1980) originally isolated AAP, a natural antiarrhythmic peptide from bovine atrium. It suppressed CaCl2induced, aconitidine-induced and ouabain-induced ventricular arrhythmias in mice (Aonuma et al. 1983). Several derivatives were synthesised. AAP10 increased the dispersion of action potential duration during regional ischaemia in rabbit hearts without significantly changing heart rate, myocardial contractility, action potential duration and morphology, effective refractory period and mean coronary flow (Dhein et al. 1994). It was also shown to increase gap junction conductance in guinea pig cardiomyocytes and was investigated in a healed rabbit infarct model (Dhein et al. 2001; Ren et al. 2006). Significant reductions in VT inducibility were observed and, in the long QT rabbit model, transmural dispersion of repolarisation and torsades-de-pointes were suppressed (Quan et al. 2009). ZP123 (rotagaptide) is a chemically modified version of AAP10 and has been extensively studied in animal models and early phase clinical trials. It has highly specific effects on Cx43 as opposed to Cx26, Cx32 or Cx40. Good evidence has been presented that it increases myocardial conductivity in rabbit models and the inducibility of VT in a healed infarct model. Indeed, in guinea pig hearts, it reduces the dispersion of repolarisation during acute ischaemic conditions. Furthermore, it prevents the ischaemia-induced slowing of conduction velocity in isolated guinea pig hearts, VT induction during coronary artery ligation in canine models and reperfusion arrhythmias (Xing et al. 2003). Rotagaptide has been studied in two phase 1 single-dose or double-blinded randomised controlled trials. Its use is restricted to in-hospital

Cell Tissue Res

intravenous administration and, since it is cleared through the kidney, it should possibly be restricted in patients with renal impairment. Although it has been studied in phase II trials for patients with unstable angina, further trials have not proceeded (Kjolbye et al. 2007). Second-generation agents include GAP134 and RXP-E, which stabilises Cx43 in acidotic conditions. GAP-134 has been shown to decrease atrial effective refractory period in a pacing model of atrial fibrillation (AF), although this effect appears unrelated to Cx40 and Cx43 mRNA levels or spatial Cx43 distribution (Laurent et al. 2009). Phase 1 trials of GAP-134, which can be orally administered, have not shown any adverse effects on haematological, biochemical or electrocardiogram (ECG) parameters in healthy volunteers. No data are available at present from examination of its efficacy as an anti-arrhythmic in man. Theoretical concerns have been raised that hemichannel opening induced by these agents might promote cellular ATP loss and volume overload leading to cell death. Hemichannels are generally not considered to be permeable under physiological conditions but, during ischaemia, they might open allowing large inward cellular fluxes of Na+ and Ca2+ and the loss of metabolites in addition to ATP (Saez et al. 2010). Interestingly, this effect can be prevented by a short peptide derived from the sequence of the intracellular loop of Cx43 (Wang et al. 2013). The utilisation of these agents should also be taken in context whereby fibrosis might be more important in promoting reductions in conduction reserve and, therefore, gap junction openers will have limited effects. In a related but distinct approach, a cell-permeable peptide mimetic of the C-terminus of Cx43 was synthesised that inhibited interaction with zonula occludens-1. In an in vivo model of the ischaemic border zone, application of the peptide prevented the lateralisation of Cx43, improved ventricular depolarisation dynamics and reduced the incidence of inducible ventricular arrhythmias (O’Quinn et al. 2011).

Connexin abnormalities in cardiac disease Arrhythmogenic right ventricular cardiomyothy Studies of the structural pathology of Carvajal syndrome (an ARVC autosomal recessive variant) have shown the abnormal structure of intercalated discs, with reduced amounts of desmoplakin and plakoglobin (Kaplan et al. 2004a). This results in a redistribution of Cx43 such that it is less well localised to the intercalated disc. The examination of patients with Naxos disease (an autosomal recessive form of ARVC) has also shown that plakoglobin fails to locate at intercalated discs and to form normal gap junctions, despite normal intracellular levels and desmoplakin and N-cadherin both being present (Kaplan et al. 2004b). These observations, together with those from animal studies (see above), indicate a complex interplay

of structural pathology and slowed conduction attributable to the abnormal trafficking and function of Cx43 and SCN5A at the intercalated disc as the determinant of the arrhythmic phenotype. Ischaemic heart disease As has been recognised for many years, complex fractionated electrograms are recorded in diseased and failing hearts and these patients have significantly increased risks of lethal ventricular arrhythmias (de Bakker et al. 1993). Histological studies have revealed complex areas of fibrosis interdigitated with myocardial bundles in the infarct border zone and decreased expression and mislocalisation of Cx43 at sites of reentrant circuits in this region (Peters et al. 1997). In a rat model of both diabetes mellitus and hypertension, downregulation and/or the abnormal distribution of myocardial Cx43-positive gap junctions is associated with increased susceptibility to hypokalaemia-induced VF (Okruhlicova et al. 2002; Tribulova et al. 2003; Fialova et al. 2008). Myocardial gap junction remodelling has also been associated with increased vulnerability to VF in hypertriglyceridaemic rats (Zicha et al. 2006). Perfusion of the heart with hypokalaemic solution causes an increase in intracellular Ca2+ concentration. Diabetic or hypertensive rat hearts with abnormal Ca2+ handling are also more prone to develop premature ventricular beats compared with controls (Tribulova et al. 2003). Furthermore, acute Ca2+ overload might contribute to disturbances in coordinated contraction probably attributable to cell-to-cell uncoupling (de Groot and Coronel 2004; Tribulova et al. 2003) leading to differences in mechanical strain and changes in action potential duration through mechano-electric feedback and activation of stretch-activated ion channels (de Groot and Coronel 2004). This again creates inhomogeneity in conduction and repolarisation promoting re-entrant VT and wavebreak with the initiation of VF. Therefore, gap junction remodelling and redistribution in the ischaemic or infarcted myocardium can induce direct and downstream effects on the myocardial substrate to create a highly arrhythmogenic environment. During ischaemia, pathological decreases in the conductance of gap junctions occur following increases in intracellular Ca2+ concentration (Smith et al. 1995; de Groot et al. 2001) and intracellular acidification (Yan and Kleber 1992) and through changes in catecholamine-induced increases in cellular cAMP, which in turn modulate levels of phosphorylation. Acute increases in intracellular Ca2+ concentration occur in ischaemic rabbit models leading to gap junctional uncoupling and decreased conductance (Dekker et al. 1996; Smith et al. 1995; Gutstein et al. 2001). This results in conduction slowing and blockage that is exacerbated by increases in intracellular pH (Kleber et al. 1986). Myocardial ischaemia also causes Cx43 rapidly to dephosphorylate leading to its lateralisation

Cell Tissue Res

and transfer from the intercalated discs to intracellular pools and to electrical uncoupling (Beardslee et al. 2000; MarquezRosado et al. 2012). Dephosphorylation of connexins might also be involved in the lateralisation of gap junctions resulting in conduction abnormalities in AF (Dobrev et al. 2012). Atrial fibrillation The pathophysiology of AF is complex, consisting of triggered events arising from the pulmonary and thoracic veins, in its early phases, and leading to both electrical remodelling and structural changes in the atrial myocardium as it is impacted by aging, hypertension and haemodynamic stresses promoting stretch and fibrosis. Therefore, the atria become more arrhythmogenic because of reduced effective refractory period, enhanced spatial dispersion of refractoriness or abnormal atrial impulse conduction initiating and maintaining AF (Nattel 2002). The principal atrial connexin, Cx40, has been linked to AF. Several studies in Cx40 knockout mice have indicated that Cx40 is the dominant isoform for impulse conduction in the atria and the conduction system. Most of these studies have demonstrated that full deficiency for Cx40 prolongs the P-wave, PQ/PR interval, QRS and QTc on surface ECG and that the mice are susceptible to atrial tachyarrhythmias (Simon et al. 1998; Bagwe et al. 2005; Hagendorff et al. 1999). Epicardial mapping has revealed that the prolonged P-wave and PQ/PR-interval are attributable to reduced conduction velocity in the atria (Verheule et al. 1999). The goat model of AF represents one of the best characterised animal models. Induction of persistent AF by high rate burst pacing leads to the heterogeneous spatial distribution of Cx40, whereas the expression of Cx43 remains unchanged (van der Velden et al. 1998); patches of cells virtually devoid of Cx40 lie adjacent to populations with almost normal expression (van der Velden et al. 2000). Interestingly, reverse remodelling can occur with the termination of AF, leading to a gradual normalisation of the gap junction distribution pattern and Cx40 expression (Ausma et al. 2003). Total Cx40 protein levels are unchanged or reduced in long-lasting AF with unaltered levels of Cx40 mRNA (van der Velden et al. 1998; Thijssen et al. 2002). Several human studies have investigated connexin expression and distribution during AF. During sinus rhythm, gap junctions are expressed mainly at the terminal end of the cells, although side-to-side distributions can also be seen commonly in the atria (Polontchouk et al. 2001; Dupont et al. 2001; Kostin et al. 2002; Dhein et al. 2008). However, with the development of AF, Cx40 expression changes to become predominantly side-to-side and heterogeneous, although the expression of N-cadherin and desmoplakin remains normal (Polontchouk et al. 2001; Dupont et al. 2001; Kostin et al. 2002; Li et al. 2009; Takeuchi et al. 2006). In patients, with ischaemic heart disease and no history of arrhythmias, postoperative AF is more likely when levels of Cx40 protein are

relatively high prior to surgery (Dupont et al. 2001). Other studies, however, which have investigated changes in Cx40 expression and distribution in patients with long-lasting AF (at least 3 months and thus following atrial remodelling), have shown inconsistent results with respect to the amount of Cx40 protein level during AF. Some of these studies have demonstrated that Cx40 protein levels are increased with lateralised expression in the atria, independent of AF aetiology (Polontchouk et al. 2001; Wetzel et al. 2005). Others have found that the expression of Cx40 during AF is significantly reduced (Kostin et al. 2002; Nao et al. 2003; Wilhelm et al. 2006), whereas some studies have found no differences (Li et al. 2009; Takeuchi et al. 2006) or that Cx40 expression levels are dependent on the extracellular Ca2+ level (Dhein et al. 2008). Some of the differences in Cx40 distribution might be attributable to methodological differences in the antibodies employed to detect the various connexin proteins. Two mechanisms by which Cx40 plays a role in triggering AF from pulmonary venous sleeves are possible: (1) the pulmonary vein contains spontaneously rhythmic cells that share a sinus nodal-like gap junction expression and these might act as automatic foci; (2) abnormal and discontinuous gap junction expression with rapid changes in fibre direction might facilitate micro-re-entry resulting in the pre-excitatory triggering of the atrial myocardium. Inherited differences in the Cx40 gene (GJA5) have been associated with atrial arrhythmias. Groenewegen et al. (2003) have shown that atrial standstill is caused by a rare polymorphism of the promoter of the Cx40 gene at nucleotides −44 (G→A) and +71 (A→G). This occurs in 7 % of the population but only when expressed in combination with a novel mutation in the sodium channel gene SCN5A (Groenewegen et al. 2003). Firouzi et al. (2004) have correlated vulnerability for AF to this Cx40 promoter polymorphism in patients without structural heart disease in the absence of atrial remodelling. They have compared the electrophysiological characteristics of 30 patients with supraventricular tachycardia and very rare episodes of AF with those free of AF. They have illustrated that homozygous carriers of the minor haplotype (−44AA/+71GG) are more prone to both the inducibility of AF by programmed electrical stimulation and the spontaneous occurrence of AF episodes. This predisposition to initiation of AF appears to be related to the enhanced dispersion of atrial refractoriness (Firouzi et al. 2004). To what extent the level or distribution of Cx40 protein is decreased because of this polymorphism is unclear, although the promoter genotypes do indeed affect the expression of reporter constructs. Finally, of 15 patients who exhibited idiopathic AF with early onset (±45 years) and were refractory to pharmacological therapy, four possessed one germline, four novel and three somatic heterozygous mutations in the Cx40 gene (Gollob et al. 2006).

Cell Tissue Res

Concluding remarks Significant interplay occurs between the proteins involved in structural integrity at the intercalated disc and the connexins and ion channels that determine excitability in the heart. At the fundamental level, an understanding of the cell biology and interacting proteins that determine these interdependencies will be important. The derangements in the localisation, trafficking and phosphorylation state of connexins are evident in a number of pro-arrhythmic conditions and disorders, especially ischaemic heart disease and ARVC. Furthermore, the direct or indirect modulation of the function of gap junctions might be important therapeutically and might alleviate abnormal heart rhythm in a number of cardiac diseases. However, additional work is required to investigate connexin interactions with other ion channels including the voltage-gated sodium channel, SCN5A, which could form the target of alternative anti-arrhythmic agents.

References Aonuma S, Kohama Y, Akai K, Komiyama Y, Nakajima S, Wakabayashi M, Makino T (1980) Studies on heart. XIX. Isolation of an atrial peptide that improves the rhythmicity of cultured myocardial cell clusters. Chem Pharm Bull (Tokyo) 28:3332–3339 Aonuma S, Kohama Y, Makino T, Yoshitake I, Hattori K, Morikawa K, Watanabe Y (1983) Studies on heart. XXIII. Distribution of [1-14C] acetamidino-antiarrhythmic peptide (14C-AAP) in mice. Chem Pharm Bull (Tokyo) 31:612–619 Arking DE, Pfeufer A, Post W, Kao WH, Newton-Cheh C, Ikeda M, West K, Kashuk C, Akyol M, Perz S, Jalilzadeh S, Illig T, Gieger C, Guo CY, Larson MG, Wichmann HE, Marban E, O’Donnell CJ, Hirschhorn JN, Kaab S, Spooner PM, Meitinger T, Chakravarti A (2006) A common genetic variant in the NOS1 regulator NOS1AP modulates cardiac repolarization. Nat Genet 38:644–651 Asimaki A, Kapoor S, Plovie E, Karin AA, Adams E, Liu Z, James CA, Judge DP, Calkins H, Churko J, Wu JC, MacRae CA, Kleber AG, Saffitz JE (2014) Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy. Sci Transl Med 6:240ra74 Ausma J, Velden HM van der, Lenders MH, Ankeren EP van, Jongsma HJ, Ramaekers FC, Borgers M, Allessie MA (2003) Reverse structural and gap-junctional remodeling after prolonged atrial fibrillation in the goat. Circulation 107:2051–2058 Bagwe S, Berenfeld O, Vaidya D, Morley GE, Jalife J (2005) Altered right atrial excitation and propagation in connexin40 knockout mice. Circulation 112:2245–2253 Bakker JM de, Capelle FJ van, Janse MJ, Tasseron S, Vermeulen JT, Jonge N de, Lahpor JR (1993) Slow conduction in the infarcted human heart. “Zigzag” course of activation. Circulation 88: 915–926 Beardslee MA, Laing JG, Beyer EC, Saffitz JE (1998) Rapid turnover of connexin43 in the adult rat heart. Circ Res 83:629–635 Beardslee MA, Lerner DL, Tadros PN, Laing JG, Beyer EC, Yamada KA, Kleber AG, Schuessler RB, Saffitz JE (2000) Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia. Circ Res 87:656–662

Beyer EC, Paul DL, Goodenough DA (1987) Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105:2621–2629 Cerrone M, Noorman M, Lin X, Chkourko H, Liang FX, Nagel R van der, Hund T, Birchmeier W, Mohler P, Veen TA van, Rijen HV van, Delmar M (2012) Sodium current deficit and arrhythmogenesis in a murine model of plakophilin-2 haploinsufficiency. Cardiovasc Res 95:460–468 Coppen SR, Severs NJ, Gourdie RG (1999) Connexin45 (alpha 6) expression delineates an extended conduction system in the embryonic and mature rodent heart. Dev Genet 24:82–90 Danik SB, Liu F, Zhang J, Suk HJ, Morley GE, Fishman GI, Gutstein DE (2004) Modulation of cardiac gap junction expression and arrhythmic susceptibility. Circ Res 95:1035–1041 Danik SB, Rosner G, Lader J, Gutstein DE, Fishman GI, Morley GE (2008) Electrical remodeling contributes to complex tachyarrhythmias in connexin43-deficient mouse hearts. FASEB J 22:1204–1212 Davis LM, Kanter HL, Beyer EC, Saffitz JE (1994) Distinct gap junction protein phenotypes in cardiac tissues with disparate conduction properties. J Am Coll Cardiol 24:1124–1132 Dekker LR, Fiolet JW, VanBavel E, Coronel R, Opthof T, Spaan JA, Janse MJ (1996) Intracellular Ca2+, intercellular electrical coupling, and mechanical activity in ischemic rabbit papillary muscle. Effects of preconditioning and metabolic blockade. Circ Res 79:237–246 Delmar M, McKenna WJ (2010) The cardiac desmosome and arrhythmogenic cardiomyopathies: from gene to disease. Circ Res 107: 700–714 Derksen R, Rijen HV van, Wilders R, Tasseron S, Hauer RN, Rutten WL, Bakker JM de (2003) Tissue discontinuities affect conduction velocity restitution: a mechanism by which structural barriers may promote wave break. Circulation 108:882–888 Dhein S, Manicone N, Muller A, Gerwin R, Ziskoven U, Irankhahi A, Minke C, Klaus W (1994) A new synthetic antiarrhythmic peptide reduces dispersion of epicardial activation recovery interval and diminishes alterations of epicardial activation patterns induced by regional ischemia. a mapping study. Naunyn Schmiedebergs Arch Pharmacol 350:174–184 Dhein S, Weng S, Grover R, Tudyka T, Gottwald M, Schaefer T, Polontchouk L (2001) Protein kinase Calpha mediates the effect of antiarrhythmic peptide on gap junction conductance. Cell Commun Adhes 8:257–264 Dhein S, Duerrschmidt N, Scholl A, Boldt A, Schulte JS, Pfannmuller B, Rojas-Gomez D, Scheffler A, Haefliger JA, Doll N, Mohr FW (2008) A new role for extracellular Ca2+ in gap-junction remodeling: studies in humans and rats. Naunyn Schmiedebergs Arch Pharmacol 377:125–138 Dobrev D, Carlsson L, Nattel S (2012) Novel molecular targets for atrial fibrillation therapy. Nat Rev Drug Discov 11:275–291 Dupont E, Ko Y, Rothery S, Coppen SR, Baghai M, Haw M, Severs NJ (2001) The gap-junctional protein connexin40 is elevated in patients susceptible to postoperative atrial fibrillation. Circulation 103: 842–849 Eckardt D, Theis M, Degen J, Ott T, Rijen HV van, Kirchhoff S, Kim JS, Bakker JM de, Willecke K (2004) Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion. J Mol Cell Cardiol 36:101–110 Fialova M, Dlugosova K, Okruhlicova L, Kristek F, Manoach M, Tribulova N (2008) Adaptation of the heart to hypertension is associated with maladaptive gap junction connexin-43 remodeling. Physiol Res 57:7–11 Firouzi M, Ramanna H, Kok B, Jongsma HJ, Koeleman BP, Doevendans PA, Groenewegen WA, Hauer RN (2004) Association of human connexin40 gene polymorphisms with atrial vulnerability as a risk factor for idiopathic atrial fibrillation. Circ Res 95:e29–e33 Gard JJ, Yamada K, Green KG, Eloff BC, Rosenbaum DS, Wang X, Robbins J, Schuessler RB, Yamada KA, Saffitz JE (2005)

Cell Tissue Res Remodeling of gap junctions and slow conduction in a mouse model of desmin-related cardiomyopathy. Cardiovasc Res 67:539–547 Gollob MH, Jones DL, Krahn AD, Danis L, Gong XQ, Shao Q, Liu X, Veinot JP, Tang AS, Stewart AF, Tesson F, Klein GJ, Yee R, Skanes AC, Guiraudon GM, Ebihara L, Bai D (2006) Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation. N Engl J Med 354:2677–2688 Gomes J, Finlay M, Ahmed AK, Ciaccio EJ, Asimaki A, Saffitz JE, Quarta G, Nobles M, Syrris P, Chaubey S, McKenna WJ, Tinker A, Lambiase PD (2012) Electrophysiological abnormalities precede overt structural changes in arrhythmogenic right ventricular cardiomyopathy due to mutations in desmoplakin-A combined murine and human study. Eur Heart J 33:1942–1953 Gourdie RG, Green CR, Severs NJ (1991) Gap junction distribution in adult mammalian myocardium revealed by an antipeptide antibody and laser scanning confocal microscopy. J Cell Sci 99:41–55 Groenewegen WA, Firouzi M, Bezzina CR, Vliex S, Langen IM van, Sandkuijl L, Smits JP, Hulsbeek M, Rook MB, Jongsma HJ, Wilde AA (2003) A cardiac sodium channel mutation cosegregates with a rare connexin40 genotype in familial atrial standstill. Circ Res 92: 14–22 Groot JR de, Coronel R (2004) Acute ischemia-induced gap junctional uncoupling and arrhythmogenesis. Cardiovasc Res 62:323–334 Groot JR de, Wilms-Schopman FJ, Opthof T, Remme CA, Coronel R (2001) Late ventricular arrhythmias during acute regional ischemia in the isolated blood perfused pig heart. Role of electrical cellular coupling. Cardiovasc Res 50:362–372 Guerrero PA, Schuessler RB, Davis LM, Beyer EC, Johnson CM, Yamada KA, Saffitz JE (1997) Slow ventricular conduction in mice heterozygous for a connexin43 null mutation. J Clin Invest 99: 1991–1998 Gutstein DE, Morley GE, Tamaddon H, Vaidya D, Schneider MD, Chen J, Chien KR, Stuhlmann H, Fishman GI (2001) Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. Circ Res 88:333–339 Gutstein DE, Liu FY, Meyers MB, Choo A, Fishman GI (2003) The organization of adherens junctions and desmosomes at the cardiac intercalated disc is independent of gap junctions. J Cell Sci 116:875–885 Hagendorff A, Schumacher B, Kirchhoff S, Luderitz B, Willecke K (1999) Conduction disturbances and increased atrial vulnerability in connexin40-deficient mice analyzed by transesophageal stimulation. Circulation 99:1508–1515 Hunter AW, Barker RJ, Zhu C, Gourdie RG (2005) Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion. Mol Biol Cell 16:5686–5698 Jansen JA, Veen TA van, Bakker JM de, Rijen HV van (2010) Cardiac connexins and impulse propagation. J Mol Cell Cardiol 48:76–82 Jansen JA, Noorman M, Musa H, Stein M, Jong S de, Nagel R van der, Hund TJ, Mohler PJ, Vos MA, Veen TA van, Bakker JM de, Delmar M, Rijen HV van (2012) Reduced heterogeneous expression of Cx43 results in decreased Nav1.5 expression and reduced sodium current that accounts for arrhythmia vulnerability in conditional Cx43 knockout mice. Heart Rhythm 9:600–607 Jeyaraman M, Tanguy S, Fandrich RR, Lukas A, Kardami E (2003) Ischemia-induced dephosphorylation of cardiomyocyte connexin43 is reduced by okadaic acid and calyculin A but not fostriecin. Mol Cell Biochem 242:129–134 Kaplan SR, Gard JJ, Carvajal-Huerta L, Ruiz-Cabezas JC, Thiene G, Saffitz JE (2004a) Structural and molecular pathology of the heart in Carvajal syndrome. Cardiovasc Pathol 13:26–32 Kaplan SR, Gard JJ, Protonotarios N, Tsatsopoulou A, Spiliopoulou C, Anastasakis A, Squarcioni CP, McKenna WJ, Thiene G, Basso C, Brousse N, Fontaine G, Saffitz JE (2004b) Remodeling of myocyte gap junctions in

arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease). Heart Rhythm 1:3–11 Kapoor A, Sekar RB, Hansen NF, Fox-Talbot K, Morley M, Pihur V, Chatterjee S, Brandimarto J, Moravec CS, Pulit SL, Pfeufer A, Mullikin J, Ross M, Green ED, Bentley D, Newton-Cheh C, Boerwinkle E, Tomaselli GF, Cappola TP, Arking DE, Halushka MK, Chakravarti A (2014) An enhancer polymorphism at the cardiomyocyte intercalated disc protein NOS1AP locus is a major regulator of the QT interval. Am J Hum Genet 94:854–869 King JH, Huang CL, Fraser JA (2013) Determinants of myocardial conduction velocity: implications for arrhythmogenesis. Front Physiol 4:154 Kirchhof P, Fabritz L, Zwiener M, Witt H, Schafers M, Zellerhoff S, Paul M, Athai T, Hiller KH, Baba HA, Breithardt G, Ruiz P, Wichter T, Levkau B (2006) Age- and training-dependent development of arrhythmogenic right ventricular cardiomyopathy in heterozygous plakoglobin-deficient mice. Circulation 114:1799–1806 Kjolbye AL, Haugan K, Hennan JK, Petersen JS (2007) Pharmacological modulation of gap junction function with the novel compound rotigaptide: a promising new principle for prevention of arrhythmias. Basic Clin Pharmacol Toxicol 101:215–230 Kleber AG, Janse MJ, Wilms-Schopmann FJ, Wilde AA, Coronel R (1986) Changes in conduction velocity during acute ischemia in ventricular myocardium of the isolated porcine heart. Circulation 73: 189–198 Kostin S, Klein G, Szalay Z, Hein S, Bauer EP, Schaper J (2002) Structural correlate of atrial fibrillation in human patients. Cardiovasc Res 54:361–379 Kreuzberg MM, Sohl G, Kim JS, Verselis VK, Willecke K, Bukauskas FF (2005) Functional properties of mouse connexin30.2 expressed in the conduction system of the heart. Circ Res 96:1169–1177 Kreuzberg MM, Liebermann M, Segschneider S, Dobrowolski R, Dobrzynski H, Kaba R, Rowlinson G, Dupont E, Severs NJ, Willecke K (2009) Human connexin31.9, unlike its orthologous protein connexin30.2 in the mouse, is not detectable in the human cardiac conduction system. J Mol Cell Cardiol 46:553–559 Lampe PD, TenBroek EM, Burt JM, Kurata WE, Johnson RG, Lau AF (2000) Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication. J Cell Biol 149: 1503–1512 Lampe PD, Cooper CD, King TJ, Burt JM (2006) Analysis of connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart. J Cell Sci 119:3435–3442 Laurent G, Leong-Poi H, Mangat I, Moe GW, Hu X, So PP, Tarulli E, Ramadeen A, Rossman EI, Hennan JK, Dorian P (2009) Effects of chronic gap junction conduction-enhancing antiarrhythmic peptide GAP-134 administration on experimental atrial fibrillation in dogs. Circ Arrhythm Electrophysiol 2:171–178 Li JY, Lai YJ, Yeh HI, Chen CL, Sun S, Wu SJ, Lin FY (2009) Atrial gap junctions, NF-kappaB and fibrosis in patients undergoing coronary artery bypass surgery: the relationship with postoperative atrial fibrillation. Cardiology 112:81–88 Lin R, Warn-Cramer BJ, Kurata WE, Lau AF (2001) v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication. J Cell Biol 154:815–827 Malhotra JD, Thyagarajan V, Chen C, Isom LL (2004) Tyrosinephosphorylated and nonphosphorylated sodium channel beta1 subunits are differentially localized in cardiac myocytes. J Biol Chem 279:40748–40754 Marquez-Rosado L, Solan JL, Dunn CA, Norris RP, Lampe PD (2012) Connexin43 phosphorylation in brain, cardiac, endothelial and epithelial tissues. Biochim Biophys Acta 1818:1985–1992 Miragoli M, Gaudesius G, Rohr S (2006) Electrotonic modulation of cardiac impulse conduction by myofibroblasts. Circ Res 98:801–810 Miragoli M, Salvarani N, Rohr S (2007) Myofibroblasts induce ectopic activity in cardiac tissue. Circ Res 101:755–758

Cell Tissue Res Morley GE, Vaidya D, Jalife J (2000) Characterization of conduction in the ventricles of normal and heterozygous Cx43 knockout mice using optical mapping. J Cardiovasc Electrophysiol 11:375–377 Musil LS, Cunningham BA, Edelman GM, Goodenough DA (1990) Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J Cell Biol 111:2077–2088 Naitoh K, Yano T, Miura T, Itoh T, Miki T, Tanno M, Sato T, Hotta H, Terashima Y, Shimamoto K (2009) Roles of Cx43-associated protein kinases in suppression of gap junction-mediated chemical coupling by ischemic preconditioning. Am J Physiol Heart Circ Physiol 296:H396–H403 Nao T, Ohkusa T, Hisamatsu Y, Inoue N, Matsumoto T, Yamada J, Shimizu A, Yoshiga Y, Yamagata T, Kobayashi S, Yano M, Hamano K, Matsuzaki M (2003) Comparison of expression of connexin in right atrial myocardium in patients with chronic atrial fibrillation versus those in sinus rhythm. Am J Cardiol 91:678–683 Nattel S (2002) New ideas about atrial fibrillation 50 years on. Nature 415:219–226 O’Quinn MP, Palatinus JA, Harris BS, Hewett KW, Gourdie RG (2011) A peptide mimetic of the connexin43 carboxyl terminus reduces gap junction remodeling and induced arrhythmia following ventricular injury. Circ Res 108:704–715 Okruhlicova L, Tribulova N, Misejkova M, Kucka M, Stetka R, Slezak J, Manoach M (2002) Gap junction remodelling is involved in the susceptibility of diabetic rats to hypokalemia-induced ventricular fibrillation. Acta Histochem 104:387–391 Peters NS, Coromilas J, Severs NJ, Wit AL (1997) Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia. Circulation 95:988–996 Polontchouk L, Haefliger JA, Ebelt B, Schaefer T, Stuhlmann D, Mehlhorn U, Kuhn-Regnier F, De Vivie ER, Dhein S (2001) Effects of chronic atrial fibrillation on gap junction distribution in human and rat atria. J Am Coll Cardiol 38:883–891 Quan XQ, Bai R, Lu JG, Patel C, Liu N, Ruan Y, Chen BD, Ruan L, Zhang CT (2009) Pharmacological enhancement of cardiac gap junction coupling prevents arrhythmias in canine LQT2 model. Cell Commun Adhes 16:29–38 Reaume AG, De Sousa PA, Kulkarni S, Langille BL, Zhu D, Davies TC, Juneja SC, Kidder GM, Rossant J (1995) Cardiac malformation in neonatal mice lacking connexin43. Science 267:1831–1834 Ren Y, Zhang CT, Wu J, Ruan YF, Pu J, He L, Wu W, Chen BD, Wang WG, Wang L (2006) The effects of antiarrhythmic peptide AAP10 on ventricular arrhythmias in rabbits with healed myocardial infarction. Zhonghua Xin Xue Guan Bing Za Zhi 34:825–828 Rhett JM, Jourdan J, Gourdie RG (2011) Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1. Mol Biol Cell 22:1516–1528 Rhett JM, Veeraraghavan R, Poelzing S, Gourdie RG (2013) The perinexus: sign-post on the path to a new model of cardiac conduction? Trends Cardiovasc Med 23:222–228 Rijen HV van, Eckardt D, Degen J, Theis M, Ott T, Willecke K, Jongsma HJ, Opthof T, Bakker JM de (2004) Slow conduction and enhanced anisotropy increase the propensity for ventricular tachyarrhythmias in adult mice with induced deletion of connexin43. Circulation 109: 1048–1055 Rohr S, Kucera JP, Kleber AG (1998) Slow conduction in cardiac tissue. I. Effects of a reduction of excitability versus a reduction of electrical coupling on microconduction. Circ Res 83:781–794 Saez JC, Schalper KA, Retamal MA, Orellana JA, Shoji KF, Bennett MV (2010) Cell membrane permeabilization via connexin hemichannels in living and dying cells. Exp Cell Res 316:2377–2389 Saffitz JE, Davis LM, Darrow BJ, Kanter HL, Laing JG, Beyer EC (1995) The molecular basis of anisotropy: role of gap junctions. J Cardiovasc Electrophysiol 6:498–510

Saiz J, Ferrero JM Jr, Monserrat M, Ferrero JM, Thakor NV (1999) Influence of electrical coupling on early afterdepolarizations in ventricular myocytes. IEEE Trans Biomed Eng 46:138–147 Salameh A, Krautblatter S, Karl S, Blanke K, Gomez DR, Dhein S, Pfeiffer D, Janousek J (2009) The signal transduction cascade regulating the expression of the gap junction protein connexin43 by beta-adrenoceptors. Br J Pharmacol 158:198–208 Sato PY, Musa H, Coombs W, Guerrero-Serna G, Patino GA, Taffet SM, Isom LL, Delmar M (2009) Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes. Circ Res 105:523–526 Sato PY, Coombs W, Lin X, Nekrasova O, Green KJ, Isom LL, Taffet SM, Delmar M (2011) Interactions between ankyrin-G, plakophilin2, and connexin43 at the cardiac intercalated disc. Circ Res 109: 193–201 Scemes E (2012) Nature of plasmalemmal functional “hemichannels”. Biochim Biophys Acta 1818:1880–1883 Shaw RM, Fay AJ, Puthenveedu MA, Zastrow M von, Jan YN, Jan LY (2007) Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions. Cell 128: 547–560 Simon AM, Goodenough DA, Paul DL (1998) Mice lacking connexin40 have cardiac conduction abnormalities characteristic of atrioventricular block and bundle branch block. Curr Biol 8:295–298 Smith JH, Green CR, Peters NS, Rothery S, Severs NJ (1991) Altered patterns of gap junction distribution in ischemic heart disease. An immunohistochemical study of human myocardium using laser scanning confocal microscopy. Am J Pathol 139:801–821 Smith WT, Fleet WF, Johnson TA, Engle CL, Cascio WE (1995) The Ib phase of ventricular arrhythmias in ischemic in situ porcine heart is related to changes in cell-to-cell electrical coupling. Experimental Cardiology Group, University of North Carolina. Circulation 92: 3051–3060 Smyth JW, Shaw RM (2013) Autoregulation of connexin43 gap junction formation by internally translated isoforms. Cell Rep 5:611–618 Smyth JW, Hong TT, Gao D, Vogan JM, Jensen BC, Fong TS, Simpson PC, Stainier DY, Chi NC, Shaw RM (2010) Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium. J Clin Invest 120:266–279 Smyth JW, Vogan JM, Buch PJ, Zhang SS, Fong TS, Hong TT, Shaw RM (2012) Actin cytoskeleton rest stops regulate anterograde traffic of connexin 43 vesicles to the plasma membrane. Circ Res 110:978–989 Srisakuldee W, Jeyaraman MM, Nickel BE, Tanguy S, Jiang ZS, Kardami E (2009) Phosphorylation of connexin-43 at serine 262 promotes a cardiac injury-resistant state. Cardiovasc Res 83:672–681 Takeuchi S, Akita T, Takagishi Y, Watanabe E, Sasano C, Honjo H, Kodama I (2006) Disorganization of gap junction distribution in dilated atria of patients with chronic atrial fibrillation. Circ J 70:575–582 The Cardiac Arrhythmia Suppression Trial (CAST) Investigators (1989) Preliminary report: effect of encainide and flecainide on mortality in a randomized trial of arrhythmia suppression after myocardial infarction. N Engl J Med 321:406–412 The Cardiac Arrhythmia Suppression Trial (CAST) Investigators (1992) Effect of the antiarrhythmic agent moricizine on survival after myocardial infarction. N Engl J Med 327:227–233 Thijssen VL, Velden HM van der, Ankeren EP van, Ausma J, Allessie MA, Borgers M, Eys GJ van, Jongsma HJ (2002) Analysis of altered gene expression during sustained atrial fibrillation in the goat. Cardiovasc Res 54:427–437 Tribulova N, Okruhlicova L, Imanaga I, Hirosawa N, Ogawa K, Weismann P (2003) Factors involved in the susceptibility of spontaneously hypertensive rats to low K+-induced arrhythmias. Gen Physiol Biophys 22:369–382 Velden HM van der, Kempen MJ van, Wijffels MC, Zijverden M van, Groenewegen WA, Allessie MA, Jongsma HJ (1998) Altered

Cell Tissue Res pattern of connexin40 distribution in persistent atrial fibrillation in the goat. J Cardiovasc Electrophysiol 9:596–607 Velden HM van der, Ausma J, Rook MB, Hellemons AJ, Veen TA van, Allessie MA, Jongsma HJ (2000) Gap junctional remodeling in relation to stabilization of atrial fibrillation in the goat. Cardiovasc Res 46:476–486 Verheule S, Batenburg CA van, Coenjaerts FE, Kirchhoff S, Willecke K, Jongsma HJ (1999) Cardiac conduction abnormalities in mice lacking the gap junction protein connexin40. J Cardiovasc Electrophysiol 10:1380–1389 Viswanathan PC, Rudy Y (2000) Cellular arrhythmogenic effects of congenital and acquired long-QT syndrome in the heterogeneous myocardium. Circulation 101:1192–1198 Viswanathan PC, Shaw RM, Rudy Y (1999) Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence: a simulation study. Circulation 99:2466–2474 Wang N, De Vuyst E, Ponsaerts R, Boengler K, Palacios-Prado N, Wauman J, Lai CP, De Bock M, Decrock E, Bol M, Vinken M, Rogiers V, Tavernier J, Evans WH, Naus CC, Bukauskas FF, Sipido KR, Heusch G, Schulz R, Bultynck G, Leybaert L (2013) Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury. Basic Res Cardiol 108:309

Wetzel U, Boldt A, Lauschke J, Weigl J, Schirdewahn P, Dorszewski A, Doll N, Hindricks G, Dhein S, Kottkamp H (2005) Expression of connexins 40 and 43 in human left atrium in atrial fibrillation of different aetiologies. Heart 91:166–170 Wilhelm M, Kirste W, Kuly S, Amann K, Neuhuber W, Weyand M, Daniel WG, Garlichs C (2006) Atrial distribution of connexin 40 and 43 in patients with intermittent, persistent, and postoperative atrial fibrillation. Heart Lung Circ 15:30–37 Xie Y, Garfinkel A, Camelliti P, Kohl P, Weiss JN, Qu Z (2009) Effects of fibroblast-myocyte coupling on cardiac conduction and vulnerability to reentry: a computational study. Heart Rhythm 6:1641–1649 Xing D, Kjolbye AL, Nielsen MS, Petersen JS, Harlow KW, HolsteinRathlou NH, Martins JB (2003) ZP123 increases gap junctional conductance and prevents reentrant ventricular tachycardia during myocardial ischemia in open chest dogs. J Cardiovasc Electrophysiol 14:510–520 Yan GX, Kleber AG (1992) Changes in extracellular and intracellular pH in ischemic rabbit papillary muscle. Circ Res 71:460–470 Zicha J, Pechanova O, Cacanyiova S, Cebova M, Kristek F, Torok J, S i m k o F, D o b e s o v a Z , K u n e s J ( 2 0 0 6 ) H e r e d i t a r y hypertriglyceridemic rat: a suitable model of cardiovascular disease and metabolic syndrome? Physiol Res 55(Suppl 1):S49–S63