EGFR inhibition leads to enhanced desmosome assembly and cardiomyocyte cohesion via ROCK activation
Maria Shoykhet, Orsela Dervishi, Philipp Menauer, Matthias Hiermaier, Sina Moztarzadeh, Colin Osterloh, Ralf J. Ludwig, Tatjana Williams, Brenda Gerull, Stefan Kääb, Sebastian Clauss, Dominik Schüttler, Jens Waschke, Sunil Yeruva
Maria Shoykhet, Orsela Dervishi, Philipp Menauer, Matthias Hiermaier, Sina Moztarzadeh, Colin Osterloh, Ralf J. Ludwig, Tatjana Williams, Brenda Gerull, Stefan Kääb, Sebastian Clauss, Dominik Schüttler, Jens Waschke, Sunil Yeruva
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Research Article Cardiology Cell biology

EGFR inhibition leads to enhanced desmosome assembly and cardiomyocyte cohesion via ROCK activation

Abstract

Arrhythmogenic cardiomyopathy (AC) is a familial heart disease partly caused by impaired desmosome turnover. Thus, stabilization of desmosome integrity may provide new treatment options. Desmosomes, apart from cellular cohesion, provide the structural framework of a signaling hub. Here, we investigated the role of the epidermal growth factor receptor (EGFR) in cardiomyocyte cohesion. We inhibited EGFR under physiological and pathophysiological conditions using the murine plakoglobin-KO AC model, in which EGFR was upregulated. EGFR inhibition enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR and desmoglein 2 (DSG2). Immunostaining and atomic force microscopy (AFM) revealed enhanced DSG2 localization and binding at cell borders upon EGFR inhibition. Enhanced area composita length and desmosome assembly were observed upon EGFR inhibition, confirmed by enhanced DSG2 and desmoplakin (DP) recruitment to cell borders. PamGene Kinase assay performed in HL-1 cardiomyocytes treated with erlotinib, an EGFR inhibitor, revealed upregulation of Rho-associated protein kinase (ROCK). Erlotinib-mediated desmosome assembly and cardiomyocyte cohesion were abolished upon ROCK inhibition. Thus, inhibiting EGFR and, thereby, stabilizing desmosome integrity via ROCK might provide treatment options for AC.

Authors

Maria Shoykhet, Orsela Dervishi, Philipp Menauer, Matthias Hiermaier, Sina Moztarzadeh, Colin Osterloh, Ralf J. Ludwig, Tatjana Williams, Brenda Gerull, Stefan Kääb, Sebastian Clauss, Dominik Schüttler, Jens Waschke, Sunil Yeruva

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Figure 1

EGFR or SRC inhibition induces positive adhesiotropy in cardiomyocytes.

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EGFR or SRC inhibition induces positive adhesiotropy in cardiomyocytes. ...
(A) Representative Western blot showing protein expression of EGFR and PG in Jup+/+ and Jup–/– mice, NoStain Protein Labeling Reagent was used as loading control. n = 6. (B) Western blot showing expression of EGFR in patients with AC and dilated cardiomyopathy (DCM). Ponceau S staining was used as a loading control. *P ≤ 0.05 unpaired Student’s t test, n = 3 patients per group. (C) Dispase-based dissociation assay in murine cardiac slice cultures obtained from Jup+/+ and Jup–/– mice upon inhibition of EGFR or SRC. Consecutive slices were taken for controls and treatments, and treatments were normalized to the respective control slice to minimize variability due to differences in cardiac slice size. *P ≤ 0.05, 1-way ANOVA with Holm-Sidak correction, n = 7 for Jup+/+ mice and n = 6 for Jup–/– mice. (D) Dispase-based dissociation assay in HL-1 cardiomyocytes upon inhibition of EGFR or SRC by erlotinib or PP2, respectively, with representative pictures of the wells. *P ≤ 0.05, 1-way ANOVA with Holm-Sidak correction, n = 7 independent experiments. (E) Maximum projections of immunostainings in HL-1 cardiomyocytes for N-CAD and DSG2 showing an increase of DSG2 at the cell borders after erlotinib or PP2 treatments. Z-scans spanning the whole cell volume; z steps = 0.25 μm. White arrows indicate areas of increased DSG2 localization at the cell membrane. Scale bar: 10 μm. (F) Representative Western blots for immunoprecipitation of DSG2, coimmunoprecipitation of DP, EGFR, PKP2, and PG. IgG heavy chain (IgG hc) served as loading control for immunoprecipitated samples. n = 3 independent experiments.