Defining The Molecular Landscape of Reparative Cardiac Progenitor Cells by MS-Imaging and Advanced MS-Proteomics Approaches

Introduction: Understanding the importance of the epicardium in heart repair has increased the interest in developing strategies to explore its regenerative potential for human therapies. From this perspective, the development of large animal models in cardiovascular research has provided clinically relevant insights into pathophysiology, leading to significant translational models for exploring novel therapeutic strategies, among several other applications. In this context, Matrix-Assisted Laser-Desorption/Ionisation Mass Spectrometry Imaging (MALDI-MSI) has emerged as a powerful tool which provides valuable spatial information of diverse biomolecules, such as lipids, metabolites, glycans, proteins and peptides, mapped directly in situ, exploring the molecular mechanisms occurring in biological tissues. In light of this, this work primarily aims to unravel the proteomic response of novel compounds targeting the proliferation of epicardial (ECs) and epicardial-derived cells (EPDCs) through MS-based proteomic approaches. Methodology: Epicardial slices were obtained from the left ventricle of porcine hearts, cultured and treated with pharmacological compounds. Following, formalin-fixed and paraffin-embedded epicardial slices were analysed by MALDI-MSI, generating a proteomic profile of the epicardium and myocardium as regions of interest (ROIs). Complementary, nano-scale liquid chromatography-tandem mass spectrometry (nLC-MS/MS) was employed for the characterisation of the proteomic response to the pharmacological compounds. Results: Epicardial slices provided an outstanding model for studying epicardium physiology and implementing pharmacological therapy. MALDI-MSI analysis provided a region-specific proteomic profile, discriminating epicardial from myocardial zones based on the spectral profiles. Principal Component Analysis and pairwise receiver operating characteristic analysis highlighted the features with higher power of discrimination among the two groups, with AUC > 0.8, such as ions at m/z 976.50, 1019.64 and 2705.45. Furthermore, nLCMS/MS revealed the most relevant differentiating putatively identified proteins in responses to each evaluated compound, such as collagen alpha-6 (COL6A), cadherin-13 (CDH13), gap junction alpha-1 protein (GJA1), vimentin (VIM), prostaglandin E synthase-3 (PTGES3), Moesin (MSN), proliferating cell nuclear antigen (PCNA) among others. Finally, protein-protein interaction revealed Vascular Glenda Santos de Oliveira Page | iv Endothelial Growth Factor A (VEGFA) and Vascular Endothelial Growth Factor Receptor signalling, cellular response to stress, fibroblast metabolic pathways, and hypoxia-induced factor signalling as the most relevant pathways involved in those proteomic responses. Conclusions: Porcine epicardial slices proved to be a suitable organotypic model to highlight and explore molecular insights implicated in the proteomic response to novel pharmacological compounds targeting epicardium-driven heart remodelling through an advanced MS-based proteomics approach. Keywords: Proteomics; MALDI MSI; Epicardial-derived cells; Cardiovascular research.