Stabilization of cardiomyocyte adhesion via adrenergic stimulation

• Applicant: Professor Dr. Jens Waschke
• Subject Area: Anatomy and Physiology ; Nuclear Medicine, Radiotherapy, Radiobiology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 544292811

Our work for the first funding period showed that cardiomyocyte intercellular adhesion is regulated tightly and proposed that positive adhesiotropy induced by adrenergic signaling is mediated by phosphorylation of plakoglobin (Pg) at S665, which leads to strengthening of intercalated discs. This was paralleled by activation of p38MAPK and ERK which in keratinocytes regulate cell adhesion also. The impact of cardiomyocyte adhesion regulation is evident because in arrhythmogenic cardiomyopathy (AC) often mutations are found in genes coding for desmosomal components of intercalated discs including desmoglein (Dsg) 2 and Pg, which cause loss of intercellular adhesion. Since therapy of AC is limited to symptomatic approaches, an additive treatment approach focusing on stabilization of cardiomyocyte adhesion would be desirable. This is possible because we have shown recently for the desmosomal skin disease pemphigus that the phosphodiesterase-4 inhibitor apremilast is protective and stabilizes desmosomal adhesion. Our preliminary experiments indicate that apremilast induces positive adhesiotropy with Pg phosphorylation in cardiomyocytes also.Therefore, the major goal of the proposal for the second funding period is to characterize the potential of apremilast for AC treatment and to evaluate the underlying mechanisms. Besides cultured cardiomyocytes and human AC-iPSC-cardiomyocytes we will apply cardiac slice cultures and ex vivo perfused hearts from different mouse models. 1. First, we will test the effect of apremilast on cardiomyocyte adhesion using dispase-based dissociation assays. Under same conditions Confocal-, STED- and electron microscopy (TEM) will be used to characterize effects on intercalated disc structure and to get first insights into the mechanisms involved. 2. In slice cultures from a new phosphodeficient Pg-S665 model and an EPAC1-deficient model it will be clarified whether PKA-mediated Pg-S665 phosphorylation is required for strengthening of adhesion and the parallel activation of p38MAPK and ERK or whether the PKA-independent GTP exchange factor EPAC1 is required. Next, a comprehensive signaling pathway evaluation will be performed by multiplex kinase analysis, the results of which will be verified in iPSC-cardiomyocytes by Western blot analysis after Triton X protein fractionation. Inhibitor studies will reveal their functional relevance for cell adhesion. 3. Combined STED microscopy with AFM-mediated adhesion studies will delineate effects of apremilast on the molecular binding properties of Dsg2, Dsc2 and N-cadherin. 4. Finally, Langendorff-perfused hearts from the PG-deficient AC model and multi-electrode arrays (MEA) in AC-iPSC-cardiomyocytes will be used to clarify whether apremilast besides loss of cell adhesion also can ameliorate arrhythmia in AC.

• DFG Programme: Research Grants