Defining The Molecular Landscape of Reparative Cardiac Progenitor Cells by MS-Imaging and Advanced MS-Proteomics Approaches
Creators: name: Oliveira, Glenda
Publisher: name: University of Surrey
Year: 2024
Subjects: Proteomics; MALDI MSI; MS-Based Proteomics; Drug Testing; Regenerative Research; Epicardium; Epicardium-Derived Cells; Cardiovascular Research.

description: <strong style="color:rgb(47, 84, 150);">Introduction:&lt;/strong&gt; 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 organotypic models offers a more accurate representation of the complex 3D environments found in living tissues while providing clinically relevant insights into specific pathologies, such as MI. 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, proteins and peptides, mapped directly <em style="color:rgb(13, 13, 13);">in situ&lt;/em&gt;, unravelling the molecular mechanisms within biological tissues. By integrating MALDI-MSI with nanoscale liquid chromatography-tandem mass spectrometry (nLC-MS/MS) and laser capture microdissection (LCM), this study aims to untangle the proteomic response of novel compounds that target the proliferation of epicardium-derived cells (EPDCs).</em></strong><strong style="color:rgb(47, 84, 150);">Methodology:&lt;/strong&gt; Epicardial slices were obtained from the left ventricle of porcine hearts, cultured and treated with novel pharmacological compounds. Following, formalin-fixed and paraffin-embedded epicardial slices were analysed by MALDI-MSI, generating a regio-specific proteomic profile of the epicardial slices focusing on the epicardium and subepicardium area. Complementary, LMD and nLC-MS/MS were employed to characterise the epicardium and highlight proteomic changes in response to the compounds.</strong><strong style="color:rgb(47, 84, 150);">Results:&lt;/strong&gt; Porcine epicardial slices demonstrated significant potential as an ex vivo model for drug testing, effectively capturing the critical interactions at the epicardium/myocardium interface. MALDI-MSI spatially resolved the epicardium, subepicardium, and myocardium zones by identifying regio-specific molecular profiles within each area. Focusing on the epicardium, 20 &lt;em&gt;m/z&lt;/em&gt; features were identified as having high discriminative power, as indicated by receiver operating characteristic (ROC) analysis, with area under the curve (AUC) exceeding 0.9, such as &lt;em&gt;m/z&lt;/em&gt; 1522.80, &lt;em&gt;m/z&lt;/em&gt; 1105.62, &lt;em&gt;m/z&lt;/em&gt; 1121.53, and &lt;em&gt;m/z&lt;/em&gt; 1803.97. Laser capture microdissection (LMD) enabled precise extraction and characterisation of the epicardial layer, identifying 166 proteins, among them 15 proteins related to collagen-related network, including ECM1, FMOD, LOXL1, LUM, and MATN2. Finally, protein-protein interaction (PPI) network revealed that all drugs shared common mechanisms, including stress response, vascular endothelium growth factor A and vascular endothelium growth factor R2 (VEGFA-VEGR2) signalling, and metabolic pathways. However, each drug also exhibited unique mechanisms of action. Specifically, Drug 01 influenced gap junction communication and calcium regulation, Drug 02 affected the TCA cycle and HIF-1 signalling, Drug 03 uniquely targeted fibroblast metabolic pathways and Drug 04 impacted programmed cell death, the MAPK cascade, and NF-kB signalling pathways.</strong><strong style="color:rgb(47, 84, 150);">Conclusions:&lt;/strong&gt; This work represents a breakthrough by utilising epicardial slices as a model for drug testing to stimulate epicardium-derived cell proliferation. Through an MS-based proteomics approach, the spatial distribution of epicardium-specific proteins was mapped, and in-depth proteomics further revealed the complex proteomic landscape of the epicardial slices. In conclusion, this study highlights the complexity and specificity of the molecular responses elicited by four distinctive drugs, suggesting that targeted modulation of these pathways could offer substantial therapeutic benefits for treating myocardial infarction.</strong>