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 "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.

Stabilization of desmoglein-2 binding rescues arrhythmia in arrhythmogenic cardiomyopathy.

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.

Adrenergic Signaling Strengthens Cardiac Myocyte Cohesion.

RATIONALE: The sympathetic nervous system is a major mediator of heart function. Intercalated discs composed of desmosomes, adherens junctions, and gap junctions provide the structural backbone for coordinated contraction of cardiac myocytes. OBJECTIVE: Gap junctions dynamically remodel to adapt to sympathetic signaling. However, it is unknown whether such rapid adaption also occurs for the adhesive function provided by desmosomes and adherens junctions. METHODS AND RESULTS: Atomic force microscopy revealed that ß-adrenergic signaling enhances both the number of desmoglein 2-specific interactions along cell junctions and the mean desmoglein 2-mediated binding forces, whereas N-cadherin-mediated interactions were not affected. This was accompanied by increased cell cohesion in cardiac myocyte cultures and murine heart slices. Enhanced desmoglein 2-positive contacts and increased junction length as revealed by immunofluorescence and electron microscopy reflected cAMP-induced reorganization of intercellular contacts. The mechanism underlying cAMP-mediated strengthening of desmoglein 2 binding was dependent on expression of the intercalated disc plaque protein plakoglobin (Pg) and direct phosphorylation at S665 by protein kinase A: Pg deficiency as well as overexpression of the phospho-deficient Pg-mutant S665A abrogated both cAMP-mediated junctional remodeling and increase of cohesion. Moreover, Pg knockout hearts failed to functionally adapt to adrenergic stimulation. CONCLUSIONS: Taken together, we provide first evidence for positive adhesiotropy as a new cardiac function of sympathetic signaling. Positive adhesiotropy is dependent on Pg phosphorylation at S665 by protein kinase A. This mechanism may be of high medical relevance because loss of junctional Pg is a hallmark of arrhythmogenic cardiomyopathy.

Desmoglein 3-Dependent Signaling Regulates Keratinocyte Migration and Wound Healing.

The desmosomal transmembrane adhesion molecules desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3) are required for strong keratinocyte cohesion. Recently, we have shown that Dsg3 associates with p38 mitogen-activated protein kinase (p38MAPK) and suppresses its activity. Here, we further investigated the role of Dsg3-dependent control of p38MAPK function. Dsg3-deficient mice display recurrent spontaneously healing skin erosions. In lesional and perilesional biopsies, p38MAPK activation was detectable compared with control animals. This led us to speculate that Dsg3 regulates wound repair in a p38MAPK-dependent manner. Indeed, scratch-wounded keratinocyte monolayers exhibited p38MAPK activation and loss of Dsg3 in cells lining the wound edge. Human keratinocytes after silencing of Dsg3 as well as primary cells isolated from Dsg3 knockout animals exhibited accelerated migration, which was further corroborated in an ex vivo skin outgrowth assay. Importantly, migration was efficiently blocked by inhibition of p38MAPK, indicating that p38MAPK mediates the effects observed upon loss of Dsg3. In line with this, we show that levels of active p38MAPK associated with Dsc3 are increased in Dsg3-deficient cells. These data indicate that Dsg3 controls a switch from an adhesive to a migratory keratinocyte phenotype via p38MAPK inhibition. Thus, loss of Dsg3 adhesion may foster wound closure by allowing p38MAPK-dependent migration.

Novel receptor-derived cyclopeptides to treat heart failure caused by anti-ß1-adrenoceptor antibodies in a human-analogous rat model.

Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the ß1 adrenergic receptor (ß1EC2). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human ß1EC2 (amino-acids 195-225) every 4 weeks; n = 38/114 rats were control-injected with 0.9% NaCl. Intravenous application of a novel cyclic peptide mimicking ß1EC2 (ß1EC2-CP, 1.0 mg/kg every 4 weeks) or administration of the ß1-blocker bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received ß1EC2-CP/bisoprolol co-treatment. We found that ß1EC2-CP prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, ß1EC2-CP mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free anti-ß1EC2-antibodies and by targeting ß1EC2-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful anti-ß1EC2-antibodies and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to ß1-blockade represents a promising new therapeutic option in immune-mediated heart failure.

Desmoglein-2 interaction is crucial for cardiomyocyte cohesion and function.

AIMS: We determined the contribution of the desmosomal cadherin desmoglein-2 to cell-cell cohesion in cardiomyocytes. In the intercalated disc, providing mechanical strength and electrical communication between adjacent cardiomyocytes, desmoglein-2 is closely associated with N-cadherin and gap junctions. METHODS AND RESULTS: We studied intercalated discs of HL-1 cardiomyocytes by immunostaining of desmoglein-2 and N-cadherin. Cohesion was measured using a liberase-based dissociation-assay and compared with cell-free single-molecule atomic force microscopy measurements. L-tryptophan caused irregular desmoglein-2 condensation, weakened cell-cell cohesion and impaired both homophilic desmoglein-2 and N-cadherin trans-interaction, whereas l-phenylalanine had no effect. L-tryptophan did not affect N-cadherin localization and its inhibitory effect on cell-cohesion and desmoglein-2 binding, but not on N-cadherin interaction, was blocked by a desmoglein-specific tandem peptide. Moreover, Ca(2+)-depletion, desmoglein-2 knockdown, a desmoglein-specific single peptide and certain desmoglein-2 mutations associated with arrhythmogenic cardiomyopathy reduced cell-cell cohesion, whereas cell adhesion was strengthened by desmoglein-2 overexpression. Since single peptide did not interfere with N-cadherin trans-interaction, these data indicate that (i) desmoglein-2 binding is crucial for cardiomyocyte cohesion and (ii) L-tryptophan reduced both desmoglein-2 and N-cadherin binding, whereas single and tandem peptide can be used to specifically target desmoglein-2-mediated adhesion. L-tryptophan and single peptide also induced ultrastructural alterations of areae compositae. Functional analyses at the organ level revealed reduced cardiomyocyte function and inefficient response to adrenergic stimulation in both L-tryptophan- and single peptide-challenged murine Langendorff hearts paralleled by redistribution of connexin 43 in L-tryptophan-treated heart slices. CONCLUSION: Our data demonstrate that desmoglein-2 plays a critical role in cardiomyocyte cohesion and function.

Detection of anti-ß1-AR autoantibodies in heart failure by a cell-based competition ELISA.

RATIONALE: Autoantibodies directed against the second extracellular loop of the cardiac ß1-adrenergic receptor (ß1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas heart disease. Various approaches have been used to detect such autoantibodies; however, the reported prevalence varies largely, depending on the detection method used. OBJECTIVE: We analyzed sera from 167 DCM patients (ejection fraction<45%) and from 110 age-matched volunteers who did not report any heart disease themselves, with an often used simple peptide-ELISA approach, and compared it with a novel whole cell-based ELISA, using cells expressing the full transgene for the human ß1-AR. Additionally, 35 patients with hypertensive heart disease with preserved ejection fraction were investigated. METHODS AND RESULTS: The novel assay was designed according to the currently most reliable anti-TSH receptor antibody-ELISA used to diagnose Graves disease ("third-generation assay") and also detects the target antibodies by competition with a specific monoclonal anti-ß1-AR antibody (ß1-AR MAb) directed against the functionally relevant ß1-AR epitope. Anti-ß1-AR antibodies were detected in ≈60% of DCM patients and in ≈8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti-ß1-AR antibody titers (defined as inhibition of ß1-AR MAb-binding) were no longer detected after depleting sera from IgG antibodies by protein G adsorption. In contrast, a previously used ELISA conducted with a linear 26-meric peptide derived from the second extracellular ß1-AR loop yielded a high number of false-positive results precluding any specific identification of DCM patients. CONCLUSIONS: We established a simple and efficient screening assay detecting disease-relevant ß1-AR autoantibodies in patient sera yielding a high reproducibility also in high throughput screening. The assay was validated according to "good laboratory practice" and can serve as a companion biodiagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure.

FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells.

Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d.

Cardiac beta1-adrenoceptor autoantibodies in human heart disease: rationale and design of the Etiology, Titre-Course, and Survival (ETiCS) Study.

AIMS: Evidence for a pathophysiologic relevance of autoimmunity in human heart disease has substantially increased over the past years. Conformational autoantibodies stimulating the cardiac beta1-adrenoceptor (beta1-aabs) are considered of importance in heart failure development and clinical pilot studies have shown their prognostic significance in human 'idiopathic' cardiomyopathy. METHODS: We recently developed a novel highly sensitive fluorescence-based functional assay to detect stimulating beta1-aabs. We will use this method to assess Etiology, Titre-Course, and effect on Survival (ETiCS) of beta1-aabs in a prospective multicentre study with serial follow-up of patients after a first acute myocarditis or myocardial infarction. Several European core laboratories will jointly study the hypothesis that both disorders may trigger autoimmune reactions leading to the generation of beta1-aabs and/or other heart-directed aabs. Further, sera from healthy controls and well-characterized patient cohorts with dilated, ischaemic, or hypertensive cardiomyopathy will be analysed retrospectively for beta1-aab prevalence, incidence, persistence, and/or clearance. CONCLUSION: ETiCS is so far the largest clinical diagnostic study projected to address cardiac autoimmunity. It attempts to unravel the pathophysiology of cardiac autoantibody formation and persistence/clearance. ETiCS will enhance current knowledge on autoimmunity in human heart disease and promote endeavours to develop novel therapies targeting cardiac aabs.

Targeting receptor antibodies in immune cardiomyopathy.

Although autoimmunity represents a well-established pathogenetic principle in several endocrine (Graves' disease), rheumatic (systemic lupus erythematosus), and neurological disorders (myasthenia gravis, multiple sclerosis), this mechanism has only recently gained more attention in cardiac diseases. Depending on individual genetic predisposition, heart-directed autoimmune reactions are supposed to emerge as a consequence of cardiomyocyte injury induced by inflammation, ischemia, or exposure to cardiotoxic substances. Myocyte apoptosis or necrosis and subsequent liberation of a "critical amount" of cardiac autoantigens may then induce a self-directed immune response, which in the worst case results in perpetuation of autoantibody-mediated cardiac damage. In particular, functionally active autoantibodies (aabs) directed against the cardiac beta1-adrenergic receptor (beta1-aabs) have been assigned a pivotal role in the pathogenesis of immune cardiomyopathy. Conformational beta1-aabs allosterically activate the sympathetic transmembrane signaling cascade, thereby increasing sarcoplasmatic cyclic adenosine monophosphate (cAMP) and calcium concentrations. Chronic cAMP production and calcium overload are cardiotoxic, leading to myocyte apoptosis, fibrotic repair, subsequent heart muscle dysfunction, and, finally, a dilative cardiomyopathic phenotype. Elimination by (extracorporeal) immunoadsorption or direct neutralization of the harmful receptor autoantibodies in the circulating blood represent promising strategies to protect the heart from beta1-(auto)antibody-induced damage.

The fatty acid synthase of the basidiomycete Omphalotus olearius is a single polypeptide.

Fatty acids are essential components of almost all biological membranes. Additionally, they are important in energy storage, as second messengers during signal transduction, and in post-translational protein modification. De novo synthesis of fatty acids is essential for almost all organisms, and entails the iterative elongation of the growing fatty acid chain through a set of reactions conserved in all kingdoms. During our work on the biosynthesis of secondary metabolites, a 450-kDa protein was detected by SDS-PAGE of enriched fractions from mycelial lysates from the basidiomycete Omphalotus olearius. Protein sequencing of this protein band revealed the presence of peptides with homology to both alpha and beta subunits of the ascomycete fatty acid synthase (FAS) family. The FAS encoding gene of O. olearius was sequenced. The positions of its predicted 21 introns were verified. The gene encodes a 3931 amino acids single protein, with an equivalent of the ascomycetous beta subunit at the N-terminus and the a subunit at the C-terminus. This is the first report on an FAS protein from a homobasidiomycete and also the first fungal FAS which is comprised of a single polypeptide.