Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice.

BACKGROUND: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+]i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. CONCLUSIONS: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Fibroblast KATP currents modulate myocyte electrophysiology in infarcted hearts.

Cardiac metabolism remains altered for an extended period of time after myocardial infarction. Studies have shown fibroblasts from normal hearts express KATP channels in culture. It is unknown whether fibroblasts from infarcted hearts express KATP channels and whether these channels contribute to scar and border zone electrophysiology. KATP channel subunit expression levels were determined in fibroblasts isolated from normal hearts (Fb), and scar (sMI-Fb) and remote (rMI-Fb) regions of left anterior descending coronary artery (LAD) ligated rat hearts. Whole cell KATP current density was determined with patch clamp. Action potential duration (APD) was measured with optical mapping in myocyte-only cultures and heterocellular cultures with fibroblasts with and without 100 µmol/l pinacidil. Whole heart optical mapping was used to assess KATP channel activity following LAD ligation. Pinacidil activated a potassium current (35.4 ± 7.5 pA/pF at 50 mV) in sMI-Fb that was inhibited with 10 µmol/l glibenclamide. Kir6.2 and SUR2 transcript levels were elevated in sMI-Fb. Treatment with Kir6.2 short interfering RNA decreased KATP currents (87%) in sMI-Fb. Treatment with pinacidil decreased APD (26%) in co-cultures with sMI-Fb. APD values were prolonged in LAD ligated hearts after perfusion with glibenclamide. KATP channels are present in fibroblasts from the scar and border zones of infarcted hearts. Activation of fibroblast KATP channels could modulate the electrophysiological substrate beyond the acute ischemic event. Targeting fibroblast KATP channels could represent a novel therapeutic approach to modify border zone electrophysiology after cardiac injury.

Phosphatase-resistant gap junctions inhibit pathological remodeling and prevent arrhythmias.

RATIONALE: Posttranslational phosphorylation of connexin43 (Cx43) has been proposed as a key regulatory event in normal cardiac gap junction expression and pathological gap junction remodeling. Nonetheless, the role of Cx43 phosphorylation in the context of the intact organism is poorly understood. OBJECTIVE: To establish whether specific Cx43 phosphorylation events influence gap junction expression and pathological remodeling. METHODS AND RESULTS: We generated Cx43 germline knock-in mice in which serines 325/328/330 were replaced with phosphomimetic glutamic acids (S3E) or nonphosphorylatable alanines (S3A). The S3E mice were resistant to acute and chronic pathological gap junction remodeling and displayed diminished susceptibility to the induction of ventricular arrhythmias. Conversely, the S3A mice showed deleterious effects on cardiac gap junction formation and function, developed electric remodeling, and were highly susceptible to inducible arrhythmias. CONCLUSIONS: These data demonstrate a mechanistic link between posttranslational phosphorylation of Cx43 and gap junction formation, remodeling, and arrhythmic susceptibility.

Chronic Kidney Disease Induces Proarrhythmic Remodeling.

BACKGROUND: Patients with chronic kidney disease (CKD) are at increased risk of developing cardiac arrhythmogenesis and sudden cardiac death; however, the basis for this association is incompletely known. METHODS: Here, using murine models of CKD, we examined interactions between kidney disease progression and structural, electrophysiological, and molecular cardiac remodeling. RESULTS: C57BL/6 mice with adenine supplemented in their diet developed progressive CKD. Electrocardiographically, CKD mice developed significant QT prolongation and episodes of bradycardia. Optical mapping of isolated-perfused hearts using voltage-sensitive dyes revealed significant prolongation of action potential duration with no change in epicardial conduction velocity. Patch-clamp studies of isolated ventricular cardiomyocytes revealed changes in sodium and potassium currents consistent with action potential duration prolongation. Global transcriptional profiling identified dysregulated expression of cellular stress response proteins RBM3 (RNA-binding motif protein 3) and CIRP (cold-inducible RNA-binding protein) that may underlay the ion channel remodeling. Unexpectedly, we found that female sex is a protective factor in the progression of CKD and its cardiac sequelae. CONCLUSIONS: Our data provide novel insights into the association between CKD and pathologic proarrhythmic cardiac remodeling. Cardiac cellular stress response pathways represent potential targets for pharmacologic intervention for CKD-induced heart rhythm disorders.

Mice with cardiac overexpression of peroxisome proliferator-activated receptor γ have impaired repolarization and spontaneous fatal ventricular arrhythmias.

BACKGROUND: Diabetes mellitus and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electric properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation from the contribution of global metabolic defects to the increased incidence of sudden death and electric abnormalities. METHODS AND RESULTS: In order to study the effects of metabolic abnormalities on arrhythmias without the complex systemic effects of diabetes mellitus and obesity, we studied transgenic mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor γ 1 (PPARγ1) via the cardiac α-myosin heavy-chain promoter. The PPARγ transgenic mice develop abnormal accumulation of intracellular lipids and die as young adults before any significant reduction in systolic function. Using implantable ECG telemeters, we found that these mice have prolongation of the QRS and QT intervals and spontaneous ventricular arrhythmias, including polymorphic ventricular tachycardia and ventricular fibrillation. Isolated cardiomyocytes demonstrated prolonged action potential duration caused by reduced expression and function of the potassium channels responsible for repolarization. Short-term exposure to pioglitazone, a PPARγ agonist, had no effect on mortality or rhythm in WT mice but further exacerbated the arrhythmic phenotype and increased the mortality in the PPARγ transgenic mice. CONCLUSIONS: Our findings support an important link between PPARγ activation, cardiomyocyte lipid accumulation, ion channel remodeling, and increased cardiac mortality.

Enhanced fibroblast-myocyte interactions in response to cardiac injury.

RATIONALE: A critical event in the development of cardiac fibrosis is the transformation of fibroblasts into myofibroblasts. The electrophysiological consequences of this phenotypic switch remain largely unknown. OBJECTIVE: Determine whether fibroblast activation following cardiac injury results in a distinct electrophysiological phenotype that enhances fibroblast-myocyte interactions. METHODS AND RESULTS: Neonatal rat myocyte monolayers were treated with media (CM) conditioned by fibroblasts isolated from normal (Fb) and infarcted (MI-Fb) hearts. Fb and MI-Fb were also plated on top of myocyte monolayers at 3 densities. Cultures were optically mapped after CM treatment or fibroblast plating to obtain conduction velocity and action potential duration (APD(70)). Intercellular communication and connexin43 expression levels were assessed. Membrane properties of Fb and MI-Fb were evaluated using patch clamp techniques. MI-Fb CM treatment decreased conduction velocity (11.1%) compared to untreated myocyte cultures. APD(70) was reduced by MI-Fb CM treatment compared to homocellular myocyte culture (9.4%) and Fb CM treatment (6.4%). In heterocellular cultures, MI-Fb conduction velocities were different from Fb at all densities (+29.8%, -23.0%, and -16.7% at 200, 400, and 600 cells/mm(2), respectively). APD(70) was reduced (9.6%) in MI-Fb compared to Fb cultures at 200 cells/mm(2). MI-Fb had more hyperpolarized resting membrane potentials and increased outward current densities. Connexin43 was elevated (134%) in MI-Fb compared to Fb. Intercellular coupling evaluated with gap fluorescence recovery after photobleaching was higher between myocytes and MI-Fb compared to Fb. CONCLUSIONS: These data demonstrate cardiac injury results in significant electrophysiological changes that enhance fibroblast-myocyte interactions and could contribute to the greater incidence of arrhythmias observed in fibrotic hearts.

Leaky Ca2+ release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice.

The Ca2+ release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exercise-induced sudden cardiac death.

Remodeling of atrial ATP-sensitive K⁺ channels in a model of salt-induced elevated blood pressure.

Hypertension is associated with the development of atrial fibrillation; however, the electrophysiological consequences of this condition remain poorly understood. ATP-sensitive K(+) (K(ATP)) channels, which contribute to ventricular arrhythmias, are also expressed in the atria. We hypothesized that salt-induced elevated blood pressure (BP) leads to atrial K(ATP) channel activation and increased arrhythmia inducibility. Elevated BP was induced in mice with a high-salt diet (HS) for 4 wk. High-resolution optical mapping was used to measure atrial arrhythmia inducibility, effective refractory period (ERP), and action potential duration at 90% repolarization (APD(90)). Excised patch clamping was performed to quantify K(ATP) channel properties and density. K(ATP) channel protein expression was also evaluated. Atrial arrhythmia inducibility was 22% higher in HS hearts compared with control hearts. ERP and APD(90) were significantly shorter in the right atrial appendage and left atrial appendage of HS hearts compared with control hearts. Perfusion with 1 µM glibenclamide or 300 µM tolbutamide significantly decreased arrhythmia inducibility and prolonged APD(90) in HS hearts compared with untreated HS hearts. K(ATP) channel density was 156% higher in myocytes isolated from HS animals compared with control animals. Sulfonylurea receptor 1 protein expression was increased in the left atrial appendage and right atrial appendage of HS animals (415% and 372% of NS animals, respectively). In conclusion, K(ATP) channel activation provides a mechanistic link between salt-induced elevated BP and increased atrial arrhythmia inducibility. The findings of this study have important implications for the treatment and prevention of atrial arrhythmias in the setting of hypertensive heart disease and may lead to new therapeutic approaches.

The cardiac fibroblast: functional and electrophysiological considerations in healthy and diseased hearts.

Cardiac fibrosis occurs in a number of cardiovascular diseases associated with a high incidence of arrhythmias. A critical event in the development of fibrosis is the transformation of fibroblasts into an active phenotype or myofibroblast. This transformation results in functional changes including increased proliferation and changes in the release of signaling molecules and extracellular matrix deposition. Traditionally, fibroblasts have been considered to affect cardiac electrophysiology indirectly by physically isolating myocytes and creating conduction barriers. There is now increasing evidence that cardiac fibroblasts may play a direct role in modulating the electrophysiological substrate in diseased hearts. The purpose of this review is to summarize the functional changes associated with fibroblast activation, the membrane currents that have been identified in adult cardiac fibroblasts, and describe recent studies of fibroblast-myocyte electrical interactions with emphasis on the changes that occur with cardiac injury. Further analysis of fibroblast membrane electrophysiology and their interactions with myocytes will lead to a more complete understanding of the arrhythmic substrate. These studies have the potential to generate new therapeutic approaches for the prevention of arrhythmias associated with cardiac fibrosis.

Spatiotemporal electrophysiological changes in a murine ablation model.

AIMS: High recurrence rates after complex radiofrequency ablation procedures, such as for atrial fibrillation, remain a major clinical problem. Local electrophysiological changes that occur following cardiac ablation therapy are incompletely described in the literature. The purpose of this study was to determine whether alterations in conduction velocity, action potential duration (APD), and effective refractory period resolve dynamically following cardiac ablation. METHODS AND RESULTS: Lesions were delivered to the right ventricle of mice using a subxiphoid approach. The sham-operated control group (SHAM) received the same procedure without energy delivery. Hearts were isolated at 0, 1, 7, 30, and 60 days following the procedure and electrophysiological parameters were obtained using high-resolution optical mapping with a voltage-sensitive dye. Conduction velocity was significantly decreased at the lesion border in the 0, 7, and 30 day groups compared to SHAM. APD(70) at the lesion border was significantly increased at all time points compared to SHAM. Effective refractory period was significantly increased at the lesion border at 0, 1, 7, and 30 days but not at 60 days post-ablation. This study demonstrated that post-ablation electrophysiological changes take place immediately following energy delivery and resolve within 60 days. CONCLUSIONS: Cardiac ablation causes significant electrophysiological changes both within the lesion and beyond the border zone. Late recovery of electrical conduction in individual lesions is consistent with clinical data demonstrating that arrhythmia recurrence is associated with failure to maintain bi-directional conduction block.

Connexin40 imparts conduction heterogeneity to atrial tissue.

Impulse propagation in cardiac tissue is a complex process in which intercellular coupling through gap junction channels is a critical component. Connexin40 (Cx40) is an abundant gap junction protein that is expressed in atrial myocytes. Alterations in the expression of Cx40 have been implicated in atrial arrhythmogenesis. The purpose of the current study was to assess the role of Cx40 in atrial impulse propagation. High-resolution optical mapping was used to study conduction in the right and left atrial appendages of isolated Langendorff-perfused murine hearts. Wild-type (Cx40(+/+)), heterozygous (Cx40(+/-)), and knockout (Cx40(-/-)) mice, both adult and embryonic, were studied to assess the effects of reduced Cx40 expression on sinus node function and conduction velocity at different pacing cycle lengths (100 and 60 ms). In both adult and late-stage embryonic Cx40(+/+) mice, heterogeneity in CV was found between the right and left atrial appendages. Either partial (Cx40(+/-)) or complete (Cx40(-/-)) deletion of Cx40 was associated with the loss of conduction heterogeneity in both adult and embryonic mice. Additionally, sinus node impulse initiation was found to be ectopic in Cx40(-/-) mice at 15.5 days postcoitus, whereas Cx40(+/+) mice showed normal activation occurring near the crista terminalis. Our findings suggest that Cx40 plays an essential role in establishing interatrial conduction velocity heterogeneity in the murine model. Additionally, we describe for the first time a developmental requirement for Cx40 in normal sinus node impulse initiation at 15.5 days postcoitus.

Author Correction: Connexin43 expression in bone marrow derived cells contributes to the electrophysiological properties of cardiac scar tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Connexin43 expression in bone marrow derived cells contributes to the electrophysiological properties of cardiac scar tissue.

Cardiac pathologies associated with arrhythmic activity are often accompanied by inflammation. The contribution of inflammatory cells to the electrophysiological properties of injured myocardium is unknown. Myocardial scar cell types and intercellular contacts were analyzed using a three-dimensional reconstruction from serial blockface scanning electron microscopy data. Three distinct cell populations were identified: inflammatory, fibroblastic and endocardial cells. While individual fibroblastic cells interface with a greater number of cells, inflammatory cells have the largest contact area suggesting a role in establishing intercellular electrical connections in scar tissue. Optical mapping was used to study the electrophysiological properties of scars in fetal liver chimeric mice generated using connexin43 knockout donors (bmpKO). Voltage changes were elicited in response to applied current pulses. Isopotential maps showed a steeper pattern of decay with distance from the electrode in scars compared with uninjured regions, suggesting reduced electrical coupling. The tissue decay constant, defined as the distance voltage reaches 37% of the amplitude at the edge of the scar, was 0.48 ± 0.04 mm (n = 11) in the scar of the bmpCTL group and decreased 37.5% in the bmpKO group (n = 10). Together these data demonstrate inflammatory cells significantly contribute to scar electrophysiology through coupling mediated at least partially by connexin43 expression.

Reversibility of PRKAG2 glycogen-storage cardiomyopathy and electrophysiological manifestations.

BACKGROUND: PRKAG2 mutations cause glycogen-storage cardiomyopathy, ventricular preexcitation, and conduction system degeneration. A genetic approach that utilizes a binary inducible transgenic system was used to investigate the disease mechanism and to assess preventability and reversibility of disease features in a mouse model of glycogen-storage cardiomyopathy. METHODS AND RESULTS: Transgenic (Tg) mice expressing a human N488I PRKAG2 cDNA under control of the tetracycline-repressible alpha-myosin heavy chain promoter underwent echocardiography, ECG, and in vivo electrophysiology studies. Transgene suppression by tetracycline administration caused a reduction in cardiac glycogen content and was initiated either prenatally (Tg(OFF(E-8 weeks))) or at different time points during life (Tg(OFF(4-16 weeks)), Tg(OFF(8-20 weeks)), and Tg(OFF(>20 weeks))). One group never received tetracycline, expressing transgene throughout life (Tg(ON)). Tg(ON) mice developed cardiac hypertrophy followed by dilatation, ventricular preexcitation involving multiple accessory pathways, and conduction system disease, including sinus and atrioventricular node dysfunction. CONCLUSIONS: Using an externally modifiable transgenic system, cardiomyopathy, cardiac dysfunction, and electrophysiological disorders were demonstrated to be reversible processes in PRKAG2 disease. Transgene suppression during early postnatal development prevented the development of accessory electrical pathways but not cardiomyopathy or conduction system degeneration. Taken together, these data provide insight into mechanisms of cardiac PRKAG2 disease and suggest that glycogen-storage cardiomyopathy can be modulated by lowering glycogen content in the heart.

Electrical remodeling contributes to complex tachyarrhythmias in connexin43-deficient mouse hearts.

Loss of connexin43 (Cx43) gap junction channels in the heart results in a marked increase in the incidence of spontaneous and inducible polymorphic ventricular tachyarrhythmias (PVTs). The mechanisms resulting in this phenotype remain unclear. We hypothesized that uncoupling promotes regional ion channel remodeling, thereby increasing electrical heterogeneity and facilitating the development of PVT. In isolated-perfused control hearts, programmed electrical stimulation elicited infrequent monomorphic ventricular tachyarrhythmias (MVT), and dominant frequencies (DFs) during MVT were similar in the right ventricle (RV) and left ventricle (LV). Moreover, conduction properties, action potential durations (APDs), and repolarizing current densities were similar in RV and LV myocytes. In contrast, PVT was common in Cx43 conditional knockout (OCKO) hearts, and arrhythmias were characterized by significantly higher DFs in the RV compared to the LV. APDs in OCKO myocytes were significantly shorter than those from chamber-matched controls, with RV OCKO myocytes being most affected. APD shortening was associated with higher levels of sustained current in myocytes from both chambers as well as higher levels of the inward rectifier current only in RV myocytes. Thus, alterations in cell-cell coupling lead to regional changes in potassium current expression, which in this case facilitates the development of reentrant arrhythmias. We propose a new mechanistic link between electrical uncoupling and ion channel remodeling. These findings may be relevant not only in cardiac tissue but also to other organ systems where gap junction remodeling is known to occur.

Mechanistic investigation of Ca2+ alternans in human heart failure and its modulation by fibroblasts.

BACKGROUND: Heart failure (HF) is characterized, among other factors, by a progressive loss of contractile function and by the formation of an arrhythmogenic substrate, both aspects partially related to intracellular Ca2+ cycling disorders. In failing hearts both electrophysiological and structural remodeling, including fibroblast proliferation, contribute to changes in Ca2+ handling which promote the appearance of Ca2+ alternans (Ca-alt). Ca-alt in turn give rise to repolarization alternans, which promote dispersion of repolarization and contribute to reentrant activity. The computational analysis of the incidence of Ca2+ and/or repolarization alternans under HF conditions in the presence of fibroblasts could provide a better understanding of the mechanisms leading to HF arrhythmias and contractile function disorders. METHODS AND FINDINGS: The goal of the present study was to investigate in silico the mechanisms leading to the formation of Ca-alt in failing human ventricular myocytes and tissues with disperse fibroblast distributions. The contribution of ionic currents variability to alternans formation at the cellular level was analyzed and the results show that in normal ventricular tissue, altered Ca2+ dynamics lead to Ca-alt, which precede APD alternans and can be aggravated by the presence of fibroblasts. Electrophysiological remodeling of failing tissue alone is sufficient to develop alternans. The incidence of alternans is reduced when fibroblasts are present in failing tissue due to significantly depressed Ca2+ transients. The analysis of the underlying ionic mechanisms suggests that Ca-alt are driven by Ca2+-handling protein and Ca2+ cycling dysfunctions in the junctional sarcoplasmic reticulum and that their contribution to alternans occurrence depends on the cardiac remodeling conditions and on myocyte-fibroblast interactions. CONCLUSION: It can thus be concluded that fibroblasts modulate the formation of Ca-alt in human ventricular tissue subjected to heart failure-related electrophysiological remodeling. Pharmacological therapies should thus consider the extent of both the electrophysiological and structural remodeling present in the failing heart.

Abnormal conduction and morphology in the atrioventricular node of mice with atrioventricular canal targeted deletion of Alk3/Bmpr1a receptor.

BACKGROUND: The atrioventricular (AV) node is essential for the sequential excitation and optimized contraction of the adult multichambered heart; however, relatively little is known about its formation from the embryonic AV canal. A recent study demonstrated that signaling by Alk3, the type 1a receptor for bone morphogenetic proteins, in the myocardium of the AV canal was required for the development of both the AV valves and annulus fibrosus. To test the hypothesis that bone morphogenetic protein signaling also plays a role in AV node formation, we investigated conduction system function and AV node morphology in adult mice with conditional deletion of Alk3 in the AV canal. METHODS AND RESULTS: High-resolution optical mapping with correlative histological analysis of 28 mutant hearts revealed 4 basic phenotypic classes based on electrical activation patterns and volume-conducted ECGs. The frequency of AV node conduction and morphological abnormalities increased from no detectable anomalies (class I) to severe defects (class IV), which included the presence of bypass tracts, abnormal ventricular activation patterns, fibrosis of the AV node, and twin AV nodes. CONCLUSIONS: The present findings demonstrate that bone morphogenetic protein signaling is required in the myocardium of the AV canal for proper AV junction development, including the AV node.

Cardiac-specific loss of N-cadherin leads to alteration in connexins with conduction slowing and arrhythmogenesis.

The remodeling of ventricular gap junctions, as defined by changes in size, distribution, or function, is a prominent feature of diseased myocardium. However, the regulation of assembly and maintenance of gap junctions remains poorly understood. To investigate N-cadherin function in the adult myocardium, we used a floxed N-cadherin gene in conjunction with a cardiac-specific tamoxifen-inducible Cre transgene. The mutant animals appeared active and healthy until their sudden death approximately 2 months after deleting N-cadherin from the heart. Electrophysiologic analysis revealed abnormal conduction in the ventricles of mutant animals, including diminished QRS complex amplitude consistent with loss of electrical coupling in the myocardium. A significant decrease in the gap junction proteins, connexin-43 and connexin-40, was observed in N-cadherin-depleted myocytes. Perturbation of connexin function resulted in decreased ventricular conduction velocity, as determined by optical mapping. Our data suggest that perturbation of the N-cadherin/catenin complex in heart disease may be an underlying cause, leading to the establishment of the arrythmogenic substrate by destabilizing gap junctions at the cell surface.