Cardiomyocyte cohesion is increased after ADAM17 inhibition

A Disintegrin And Metalloprotease (ADAM) family proteins are involved in several cardiac diseases, and some ADAMs have been associated with cardiomyopathies. ADAM17 is known to cleave desmoglein 2 (DSG2), one of the proteins involved in the pathogenesis of arrhythmogenic cardiomyopathy (AC). Desmosomal stability is impaired in AC, an inheritable genetic disease, the underlying causes of which can be mutations in genes coding for proteins of the desmosome, such as DSG2, desmoplakin (DP), plakoglobin (PG), plakophilin 2 or desmocollin 2. Stabilizing desmosomal contacts can therefore be a treatment option. In the heart of the murine Jup−/− AC model, (Jup being the gene coding for PG) mice, elevated levels of p38MAPK, an activator of ADAM17, were found. However, ADAM17 levels were unaltered in Jup−/− mice hearts. Nonetheless, inhibition of ADAM17 led to enhanced cardiomyocyte cohesion in both Jup+/+ and Jup−/− mice, and in HL-1 cardiomyocytes. Further, enhanced cohesion in HL-1 cardiomyocytes after acute inhibition of ADAM17 was paralleled by enhanced localization of DSG2 and DP at the membrane, whereas no changes in desmosomal assembly or the desmosomal complex were observed. In conclusion, acute inhibition of ADAM17 might lead to reduced cleavage of DSG2, thereby stabilizing the desmosomal adhesion, evidenced by increased DSG2 and DP localization at cell borders and eventually cardiomyocyte cohesion. We believe that similar mechanisms exist in AC

cAMP: A master regulator of cadherin‐mediated binding in endothelium, epithelium and myocardium

Regulation of cadherin-mediated cell adhesion is crucial not only for maintaining tissue integrity and barrier function in the endothelium and epithelium but also for electromechanical coupling within the myocardium. Therefore, loss of cadherin-mediated adhesion causes various disorders, including vascular inflammation and desmosome-related diseases such as the autoimmune blistering skin dermatosis pemphigus and arrhythmogenic cardiomyopathy. Mechanisms regulating cadherin-mediated binding contribute to the pathogenesis of diseases and may also be used as therapeutic targets. Over the last 30 years, cyclic adenosine 3′,5′-monophosphate (cAMP) has emerged as one of the master regulators of cell adhesion in endothelium and, more recently, also in epithelial cells as well as in cardiomyocytes. A broad spectrum of experimental models from vascular physiology and cell biology applied by different generations of researchers provided evidence that not only cadherins of endothelial adherens junctions (AJ) but also desmosomal contacts in keratinocytes and the cardiomyocyte intercalated discs are central targets in this scenario. The molecular mechanisms involve protein kinase A- and exchange protein directly activated by cAMP-mediated regulation of Rho family GTPases and S665 phosphorylation of the AJ and desmosome adaptor protein plakoglobin. In line with this, phosphodiesterase 4 inhibitors such as apremilast have been proposed as a therapeutic strategy to stabilize cadherin-mediated adhesion in pemphigus and may also be effective to treat other disorders where cadherin-mediated binding is compromised

Adrenergic Signaling-Induced Ultrastructural Strengthening of Intercalated Discs via Plakoglobin Is Crucial for Positive Adhesiotropy in Murine Cardiomyocytes

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

The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner

Desmosomal proteins are components of the intercalated disc and mediate cardiac myocyte adhesion. Enhancement of cardiac myocyte cohesion, referred to as \textquotedblpositive adhesiotropy\textquotedbl, 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

Environmental enteropathy in undernourished Pakistani children: Clinical and histomorphometric analyses

Despite nutrition interventions, stunting thought to be secondary to underlying environmental enteropathy (EE) remains pervasive among infants residing in resource-poor countries and remains poorly characterized. From a birth cohort of 380 children, 65 malnourished infants received 12 weeks of nutritional supplementation with ready-to-use therapeutic food (RUTF). Eleven children with insufficient response to RUTF underwent upper endoscopy with duodenal biopsies, which were compared with U.S., age-matched specimens for healthy, celiac disease, non-celiac villous atrophy, non-celiac intraepithelial lymphocytosis, and graft-versus-host disease patients. Of the 11 children biopsied, EE was found in 10 (91%) with one subject with celiac disease. Morphometry demonstrated decreased villus-to-crypt (V:C) ratios in EE relative to healthy and non-celiac lymphocytosis patients. Environmental enteropathy villus volumes were significantly decreased relative to healthy controls. In EE, average CD3+ cells per 100 epithelial cells and per 1,000 µm2 of lamina propria and the number of lamina propria CD20+ B-cell aggregates were increased relative to all other groups. Our results indicate that V:C ratios are reduced in EE but are less severe than in celiac disease. Environmental enteropathy intraepithelial and lamina propria T lymphocytosis is of greater magnitude than that in celiac disease. The increases in lamina propria B and T lymphocytes suggest that non-cytolytic lymphocytic activation may be a more prominent feature of EE relative to celiac disease. These results provide new insights into shared yet distinct histological and immunological features of EE and celiac disease in children

Untersuchungen zur Rolle des Darmepithels bei chronisch entzündlichen Darmerkrankungen: Über den Einfluss von Zytokinen und Glucocorticoiden auf die Expression der Chemokine CXCL8 und CXCL10 und den NF-kB Signalweg in intestinalen Epithel-Zelllinien

Intestinale Epithelzellen werden entweder direkt durch luminale Antigene oder durch inflammatorische Signale aus der unmittelbaren Umgebung aktiviert. Durch Sekretion von Chemokinen trägt das intestinale Epithel zur mukosalen Infiltration von Entzündungszellen bei chronisch entzündlichen Darmerkrankungen (CEDE) bei. Über die intestinale Expression und Sekretion des Chemokins CXCL10 (IP-10), das bei der Rekrutierung von T-Zellen und Monozyten maßgeblich ist, ist bisher wenig bekannt. Insbesondere existieren bisher keine Daten über die Rolle von Interleukin 1β des am häufigsten exprimierten Zytokins in der Darmschleimhaut bei chronisch entzündlichen Darmerkrankungen. Das Ziel der Arbeit war die Expression und Regulation der Chemokine CXCL8 (IL-8) und CXCL10 (IP-10), (in epithelialen Zelllinien zu untesuchen durch proinflammatorischen Zytokinen IL-1β Interferon gamma und TNF-alpha). Der Transkriptionsfaktor NF-κB spielt eine tragende Rollein der Pathophysiologie der chronisch entzündlichen Darmerkrankung. Er stellt daher ein potentielles pharmakologisches Ziel in der Behandlung dieser Erkrankung dar. Ein weiteres Ziel der Arbeit bestand daher darin, die Effekte von zwei NF-κB-Inhibitoren auf die Interleukin-1-induzierte CXCL8- und CXCL10-Expression in intestinalen Epithelzellen zu untersuchen. Glukokortikoide sind potente Inhibitoren der Chemokin-Gen-Expression in Immunzellen. Bisher existieren keine Daten über den Einfluß von Glukokortikoiden auf die Chemokin-Expression in intestinalen Epithelzellen, so daß auch der Effekt von Glukokortikoiden auf die Zytokin-induzierte Expression von CXCL8 und CXCL10 untersucht wurde. Die oben genannten Daten zeigten, daß IL-1β ein starker und unmittelbarer Stimulus für die CXCL8- und CXCL10-Genexpression sowohl auf RNA- als auch Proteinebene in Darmepithelzelllinien darstellt. Die differentielle Expression von CXCL10 durch proinflammatorische Zytokine unterstreicht die Bedeutung des Zusammenspiels epithelialer Zellen und Immunzellen im Rahmen einer intestinalen Entzündungsreaktion. Interleukin-1 spielt abhängig vom zellulären Kontext unter Nutzung des NF-κB-Signalweges eine große Rolle bei der Induktion der CXCL10-Genexpression in epithelialen Zelllinien, so daß dieses Zytokin z. B. im Falle versagender Anti-TNF-Strategien ein lohnendes Ziel für die Entwicklung zukünftiger Therapieformen darstellt. Eine pharmakologische Inhibitition von NF-κB durch unspezifische Inhibitoren wie PDTC als zukünftige Therapieoption muß kritisch hinterfragt werden, da sie im hier vorliegenden Zellmodell zu einer Verstärkung der Interleukin-1-induzierten CXCL8-Genexpression führte, ein Phänomen, das möglicherweise mit einer Aktivierung alternativer inflammatorischer Signalwege gedeutet werden kann. Die Tatsache, daß Glukokortikoide offenbar nicht wie in klassischen Immunzellen zu einer Inhibitition von Zytokinen-stimulierter Chemokin-Genexpression führen, trägt möglicherweise zum Phänomen der Steroidresistenz bei Patienten mit chronisch entzündlichen Darmerkrankungen bei

Structure and regulation of desmosomes in intercalated discs: Lessons from epithelia

For electromechanical coupling of cardiomyocytes, intercalated discs (ICDs) are pivotal as highly specialized intercellular contact areas. ICD consists of adhesive contacts, such as desmosomes and adherens junctions (AJs) that are partially intermingled and thereby form an area composita to provide mechanical strength, as well as gap junctions (GJ) and sodium channels for excitation propagation. In contrast, in epithelia, mixed junctions with features of desmosomes and AJs are regarded as transitory primarily during the formation of desmosomes. The anatomy of desmosomes is defined by a typical ultrastructure with dense intracellular plaques anchoring the cadherin-type adhesion molecules to the intermediate filament cytoskeleton. Desmosomal diseases characterized by impaired adhesive and signalling functions of desmosomal contacts lead to arrhythmogenic cardiomyopathy when affecting cardiomyocytes and cause pemphigus when manifesting in keratinocytes or present as cardiocutaneous syndromes when both cell types are targeted by the disease, which underscores the high biomedical relevance of these cell contacts. Therefore, comparative analyses regarding the structure and regulation of desmosomal contacts in cardiomyocytes and epithelial cells are helpful to better understand disease pathogenesis. In this brief review, we describe the structural properties of ICD compared to epithelial desmosomes and suggest that mechanisms regulating adhesion may at least in part be comparable. Also, we discuss whether phenomena such as hyperadhesion or the bidirectional regulation of desmosomes to serve as signalling hubs in epithelial cells may also be relevant for ICD