A small molecule screen identifies novel inducers of EMT that may increase epicardium-driven repair of the heart

Abstract

Abstract Background Since the adult heart has minimal capacity to repair itself, myocardial infarction often leads to pathological remodelling and consequently the development of heart failure. Unfortunately, no therapy is available to restore damaged heart tissue. The epicardium, the outer layer of the heart, plays an important role during the development of the heart via the contribution of cells and paracrine factors to the myocardium. In this process, epicardial epithelial-to-mesenchymal transition (EMT) is an essential step. In the adult heart, developmental epicardial behaviour is recapitulated upon injury, characterized by upregulation of e.g. EMT-related genes. However, this process is less efficient than fetal activation. We hypothesize that the reparative capacity of the heart can be enhanced by optimizing the participation of epicardium to repair, particularly through increasing the occurrence of EMT. The aim of this study is to find epicardial EMT-inducing compounds using a small molecule screen which will ultimately improve epicardium-driven repair after cardiac injury. Methods Epicardial cells were derived from human heart auricles that are routinely removed during thoracic surgery. These epicardial derived cells (EPDCs) were cultured as epithelial-like cells with a cobblestone morphology in the presence of a TGFβ inhibitor. Upon stimulation with TGFβ ligand (1ng/mL), EPDCs undergo EMT, characterized by a switch from cobblestone to spindle-shaped cells and upregulation of α-Smooth Muscle Actin (αSMA). Using these cells, a phenotypic screen was performed in 384 wells format using the LOPAC1280 small molecule library. EMT was assessed by αSMA immunostaining. The screen was performed three times (2x 10 μM and 1x 5 μM) to include interpatient variability. From each screen the top 20 ranked compounds were validated in multiple primary epicardial cell isolations. Results From the 1280 tested compounds, at least 54 compounds displayed a clear upregulation of αSMA compared to the negative controls. Next, after validation in individual patient isolations, five compounds were selected for further analysis. These five compounds reproducibly induce EMT, as shown by a dose-dependent decrease of epithelial marker E-Cadherin, and an increase of mesenchymal markers (SMA, Periostin) and EMT transcription factors (Snail, Slug). The compounds were not toxic to cardiomyocytes and fibroblasts and 4 out of 5 compounds were not toxic to endothelial cells. Currently, the compounds are tested for their ability to induce invasion in ex vivo hearts. Furthermore, we will determine the effect on cardiac repair in vivo by incorporating the compounds into slowly releasing, biodegradable patches that are applied onto the damaged murine heart. Conclusion A small molecule screen revealed five novel epicardial EMT-inducing compounds that have the potential to increase the contribution of the epicardium to the repair of the heart. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Dutch Heart Foundation