BACKGROUND:Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac desmosome disease, as more than 50% of affected patients carry pathogenic variants in desmosome protein-coding genes. In this study, we focused on the role and mechanisms of pathogenic and non-pathogenic autoantibodies against intercalated disc (ICD) proteins such as desmoglein2 (DSG2) in ACM patients, healthy relatives (HR), and murine ACM models. MATERIALS AND METHODS:IgG fractions from ACM patients, HR, healthy controls, and murine ACM models were isolated. Besides ELISA and cleavage assay, dissociation assay, immunostaining, Triton-X-100 assay, Western blots, and atomic force microscopy were performed in murine cardiac slices, HL-1 cells, or induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). RESULTS:IgG fractions from ACM patients and HR, but not murine ACM model-derived or grouped healthy controls IgG (G-HC), revealed positive ICD staining. Three out of six ACM patients derived IgGs that reduced cardiomyocyte cohesion. Pathogenic autoantibodies, bound to DSG2 in healthy and ACM hiPSC-CMs, cleaved and reduced DSG2 interaction at the molecular level. We investigated GSK-3β contribution to the cardiomyocyte cohesion loss and observed GSK-3β reduced baseline cohesion in cultured cardiomyocytes and cardiac slices. Among five ACM-IgGs, three HR-IgGs tested, three pathogenic ACM-IgGs activated GSK-3β upstream of p38MAPK, leading to phosphorylation and junctional loss of β-catenin. GSK-3β inhibition rescued the loss of cell cohesion in ACM hiPSC-CMs. CONCLUSION:Pathogenic autoantibodies targeting DSG2 are present in ACM patients and impair cardiomyocyte cohesion in a GSK-3β-dependent manner. In contrast, autoantibodies are absent in murine ACM models and are non-pathogenic in some patients and HR.

Cardiac autonomic nervous system (ANS) dysregulation is a hallmark of cardiovascular diseases such as heart failure, arrhythmias and myocardial infarction. To understand the role of ANS in cardiomyocyte cohesion, we have previously identified that adrenergic signaling enhances cardiomyocyte cohesion via PKA-mediated plakoglobin phosphorylation at serine 665, which we termed as positive adhesiotropy. In this study, we investigated the role of parasympathetic nervous system in cardiomyocyte cohesion, using carbachol (CCH) as a cholinergic agonist that binds to the muscarinic receptors. Dissociation assays performed in HL-1 cells, grown in norepinephrine (NE) containing medium for baseline adrenergic stimulation, and in murine cardiac slice cultures from wild type (wt) and plakoglobin (Pg) knockout (Jup ) mice revealed an impaired cardiomyocyte cohesion after CCH treatment. Electron microscopy revealed that CCH reduced intercalated disc plaque thickness in both wt and Jup mice. Immunostaining and atomic force microscopy in HL-1 cells demonstrated that CCH reduced desmoglein 2 (Dsg2) localization and binding at cell borders. Immunoprecipitation showed no alterations in the interaction of Dsg2, desmoplakin, plakophilin 2 and Pg after CCH treatment. Nevertheless, knockdown of any of the above genes abrogated the loss of cardiomyocyte cohesion in response to CCH. In HL-1 cells, CCH inhibited ERK phosphorylation but not Pg phosphorylation at serine 665 induced by forskolin/rolipram (FR) combination used for adrenergic stimulation. In addition, CCH activated the AKT/GSK-3β axis in the presence of NE. In conclusion, our results demonstrate that cholinergic signaling antagonizes the positive effect of adrenergic signaling on cardiomyocyte cohesion and thus causes negative adhesiotropy, which is independent of Pg phosphorylation.

Arrhythmogenic cardiomyopathy (AC) is a genetic disease causing arrhythmia and sudden cardiac death with only symptomatic therapy available at present. Mutations of desmosomal proteins, including desmoglein-2 (Dsg2) and plakoglobin (Pg), are the major cause of AC and have been shown to lead to impaired gap junction function. Recent data indicated the involvement of anti-Dsg2 autoantibodies in AC pathogenesis. We applied a peptide to stabilize Dsg2 binding similar to a translational approach to pemphigus, which is caused by anti-desmoglein autoantibodies. We provide evidence that stabilization of Dsg2 binding by a linking peptide (Dsg2-LP) is efficient to rescue arrhythmia in an AC mouse model immediately upon perfusion. Dsg2-LP, designed to cross-link Dsg2 molecules in proximity to the known binding pocket, stabilized Dsg2-mediated interactions on the surface of living cardiomyocytes as revealed by atomic force microscopy and induced Dsg2 oligomerization. Moreover, Dsg2-LP rescued disrupted cohesion induced by siRNA-mediated Pg or Dsg2 depletion or l-tryptophan, which was applied to impair overall cadherin binding. Dsg2-LP rescued connexin-43 mislocalization and conduction irregularities in response to impaired cardiomyocyte cohesion. These results demonstrate that stabilization of Dsg2 binding by Dsg2-LP can serve as a novel approach to treat arrhythmia in patients with AC.

Intercalated discs (ICDs), which connect adjacent cardiomyocytes, are composed of desmosomes, adherens junctions (AJs) and gap junctions (GJs). Previous data demonstrated that adrenergic signaling enhances cardiac myocyte cohesion, referred to as positive adhesiotropy, via PKA-mediated phosphorylation of plakoglobin (PG). However, it was unclear whether positive adhesiotropy caused ultrastructural modifications of ICDs. Therefore, we further investigated the role of PG in adrenergic signaling-mediated ultrastructural changes in the ICD of cardiomyocytes. Quantitative transmission electron microscopy (TEM) analysis of ICD demonstrated that cAMP elevation caused significant elongation of area composita and thickening of the ICD plaque, paralleled by enhanced cardiomyocyte cohesion, in WT but not PG-deficient cardiomyocytes. STED microscopy analysis supported that cAMP elevation ex vivo enhanced overlap of desmoglein-2 (Dsg2) and N-cadherin (N-cad) staining in ICDs of WT but not PG-deficient cardiomyocytes. For dynamic analyses, we utilized HL-1 cardiomyocytes, in which cAMP elevation induced translocation of Dsg2 and PG but not of N-cad to cell junctions. Nevertheless, depletion of N-cad but not of Dsg2 resulted in a decrease in basal cell cohesion whereas positive adhesiotropy was abrogated in monolayers depleted for either Dsg2 or N-cad. In the WT mice, ultrastrutural changes observed after cAMP elevation were paralleled by phosphorylation of PG at serine 665. Our data demonstrate that in murine hearts adrenergic signaling enhanced N-cad and Dsg2 in the ICD paralleled by ultrastrutural strengthening of ICDs and that effects induced by positive adhesiotropy were strictly dependent on Pg.

Arrhythmogenic cardiomyopathy (AC) is a heart disease often caused by mutations in genes coding for desmosomal proteins, including desmoglein-2 (DSG2), plakoglobin (PG), and desmoplakin (DP). Therapy is based on symptoms and limiting arrhythmia, because the mechanisms by which desmosomal components control cardiomyocyte function are largely unknown. A new paradigm could be to stabilize desmosomal cardiomyocyte adhesion and hyperadhesion, which renders desmosomal adhesion independent from Ca2+. Here, we further characterized the mechanisms behind enhanced cardiomyocyte adhesion and hyperadhesion. Dissociation assays performed in HL-1 cells and murine ventricular cardiac slice cultures allowed us to define a set of signaling pathways regulating cardiomyocyte adhesion under basal and hyperadhesive conditions. Adrenergic signaling, activation of PKC, and inhibition of p38MAPK enhanced cardiomyocyte adhesion, referred to as positive adhesiotropy, and induced hyperadhesion. Activation of ERK1/2 paralleled positive adhesiotropy, whereas adrenergic signaling induced PG phosphorylation at S665 under both basal and hyperadhesive conditions. Adrenergic signaling and p38MAPK inhibition recruited DSG2 to cell junctions. In PG-deficient mice with an AC phenotype, only PKC activation and p38MAPK inhibition enhanced cardiomyocyte adhesion. Our results demonstrate that cardiomyocyte adhesion can be stabilized by different signaling mechanisms, which are in part offset in PG-deficient AC.

Arrhythmogenic cardiomyopathy (AC) is a heart disease caused by mutations in genes encoding for desmosomal proteins such as desmoglein 2 (Dsg2) and plakoglobin (Pg) and partially paralleled with skin defects. However, therapy is symptomatic to limit arrhythmia since the mechanisms by which desmosomal components control cardiomyocyte adhesion and function are largely unknown. We have previously shown that adrenergic signaling and PKC enhance cardiomyocyte adhesion, the further of which is dependent on phosphorylation of Pg at S665. Here, we define a set of signaling pathways differentially regulating cardiomyocyte adhesion under basal as well as hyper‐adhesive, i.e., Ca2+‐ independent conditions, both of which are known to be important for epidermal integrity. In cardiomyocyte cell and cardiac slice cultures, adrenergic signaling, activation of PKC and inhibition of p38MAPK enhanced cardiomyocyte adhesion, which we refer to as positive adhesiotropy, and induced hyper‐adhesion. Positive adhesiotropy was paralleled by activation of and dependent on ERK1/2, whereas Pg phosphorylation and Dsg2 recruitment to cell junctions were induced by adrenergic signaling only. In contrast, inhibition of ERK signaling did not affect hyper‐adhesion. Finally, in heart slices from mice deficient for Pg, which displayed a strong AC phenotype, PKC activation and p38MAPK inhibition but not adrenergic signaling enhanced cardiomyocyte adhesion. Taken together, these results demonstrate that cardiomyocyte adhesion is tightly regulated by signaling mechanisms, which are in part off‐set in Pg‐dependent AC.Support or Funding InformationDFG grant to Jens Waschke with grant number WA2474/11‐1

Desmosomal proteins are components of the intercalated disc and mediate cardiac myocyte adhesion. Enhancement of cardiac myocyte cohesion, referred to as “positive adhesiotropy”, was demonstrated to be a function of sympathetic signaling and to be relevant for a sufficient inotropic response. We used the inotropic agent digitoxin to investigate the link between inotropy and adhesiotropy. In contrast to wild-type hearts, digitoxin failed to enhance pulse pressure in perfused mice hearts lacking the desmosomal protein plakoglobin which was paralleled with abrogation of plaque thickening indicating that positive inotropic response requires intact desmosomal adhesion. Atomic force microscopy revealed that digitoxin increased the binding force of the adhesion molecule desmoglein-2 at cell–cell contact areas. This was paralleled by enhanced cardiac myocyte cohesion in both HL-1 cardiac myocytes and murine cardiac slices as determined by dissociation assays as well as by accumulation of desmosomal proteins at cell–cell contact areas. However, total protein levels or cytoskeletal anchorage were not affected. siRNA-mediated depletion of desmosomal proteins abrogated increase of cell cohesion demonstrating that intact desmosomal adhesion is required for positive adhesiotropy. Mechanistically, digitoxin caused activation of ERK1/2. In line with this, inhibition of ERK1/2 signaling abrogated the effects of digitoxin on cell–cell adhesion and desmosomal reorganization. These results show that the positive inotropic agent digitoxin enhances cardiac myocyte cohesion with reorganization of desmosomal proteins in an ERK1/2-dependent manner. Desmosomal adhesion seems to be important for a sufficient positive inotropic response of digitoxin treatment, which can be of medical relevance for the treatment of heart failure.