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.
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.
Our data demonstrate that desmoglein-2 plays a critical role in cardiomyocyte cohesion and function.
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 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 to 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.Journal of Investigative Dermatology accepted article preview online, 30 September 2015. doi:10.1038/jid.2015.380.
AimsEvidence for a pathophysiologic relevance of autoimmunity in human heart disease has substantially increased over the past years. Conformational autoantibodies stimulating the cardiac b1-adrenoceptor (b1-aabs) are considered of importance in heart failure development and clinical pilot studies have shown their prognostic significance in human 'idiopathic' cardiomyopathy. MethodsWe recently developed a novel highly sensitive fluorescence-based functional assay to detect stimulating b1-aabs. We will use this method to assess Etiology, Titre-Course, and effect on Survival (ETiCS) of b1-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 b1-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 b1-aab prevalence, incidence, persistence, and/or clearance. ConclusionETiCS 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.--
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 β1 adrenergic receptor (β1EC2). 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 β1EC2 (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 β1EC2 (β1EC2-CP, 1.0 mg/kg every 4 weeks) or administration of the β1-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 β1EC2-CP/bisoprolol co-treatment. We found that β1EC2-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, β1EC2-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-β1EC2-antibodies and by targeting β1EC2-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-β1EC2-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 β1-blockade represents a promising new therapeutic option in immune-mediated heart failure.
Desmosomal proteins including desmoglein‐2 (DSG2), plakoglobin (PG), and desmoplakin (DP) are components of the intercalated disc and mediate cardiac myocyte cell‐cell adhesion. Enhancement of cardiac myocyte cohesion, referred to as “positive adhesiotropy”, was demonstrated to be a function of sympathetic signaling in the heart and to be relevant for a sufficient positive inotropic response to adrenergic stimulation. Here we used the positive inotropic agent digitoxin to further investigate the link between inotropy and adhesiotropy in cardiac myocytes. Digitoxin failed to enhance cardiac contractility in Langendorff‐perfused mouse hearts lacking the desmosomal plaque protein PG indicating that a positive inotropic response requires intact desmosomal adhesion. To investigate a potential effect of digitoxin on desmosomal adhesion, atomic force microscopy was applied and revealed that digitoxin increased the binding force of the adhesion molecule DSG2 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 DSG2, DP and PG at cell‐cell contact areas. However, total protein levels or cytoskeletal anchorage of desmosomal proteins was not affected. siRNA‐mediated depletion of DSG2, PG or DP abrogated increase of cell cohesion demonstrating that intact desmosomal adhesion is required for positive adhesiotropy. Mechanistically, digitoxin caused activation of ERK1/2 but not of p38MAPK or Src. In line with this, inhibition of ERK1/2 signaling using the inhibitor UO 126 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 medical relevant for the treatment of heart failure. Support or Funding Information This work was supported by the Ludwig‐Maximilians‐Universität Munich with the Wifomed program and the Deutsche Forschungsgemeinschaft DFG [grant number WA2474/11‐1 to J.W.] This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rationale: Autoantibodies directed against the second extracellular loop of the cardiac 1-adrenergic receptor (1-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 1-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-1-AR antibody (1-AR MAb) directed against the functionally relevant 1-AR epitope. Anti-1-AR antibodies were detected in Ϸ60% of DCM patients and in Ϸ8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti-1-AR antibody titers (defined as inhibition of 1-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 1-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 1-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. (Circ Res. 2012;111:675-684.)
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