INTRODUCTION: The epicardium provides progenitor cells and paracrine signals crucial for cardiac development and repair (1) . Despite its reported dormant status in adulthood, the adult epicardium reactivates in response to cardiac damage to provide pro-reparatory signals, influencing both cardiomyocytes and endothelial cells (1) . Enhancing the reparative potential of the epicardium represents an interesting novel route of intervention, but the paucity of adult in vitro epicardial models hampers progress (2). Advancing models that integrate epicardial cells with other cardiac cell types will be critical for dissecting cell-cell crosstalk in adults in both homeostasis and disease.
AIMS: Develop a 3D epicardial model, characterise this model using a variety of imaging techniques.
METHODS: This project engineered a multicellular heart spheroid model inclusive of the epicardial layer. Cardioids were formed with cardiomyocytes (rat neonatal or H9C2), fibroblasts (rat neonatal or human), and human endothelial cells in a 12,000:12,000:12,000 ratio. Two epicardioids models were developed that consisted of either 1,000 adult pig epicardial cells (EPDCs) or mouse embryonic epicardial cells (MECs). These ratios were chosen to mimic the in vivo composition. Spheroids were optimized for cell ratio and culture conditions and subsequently characterized via bright field and confocal microscopy. Structural organisation of cell types was determined using immunostaining (CD31,MSLN and connexin-43) and viability was assessed with both live dead staining and metabolic activity assays. To determine activity of cells within the spheroids they were challenged with pro-fibrotic TGF-β1 and hypertrophy stimuli such as phenylephrine (PH) and outcomes were assessed via immunostaining and qPCR.
RESULTS: Spheroids formed most robustly at 1:1:1 ratio of rat fibroblasts and cardiomyocytes, and human endothelial cells in cardiomyocyte media yielded spheroids with no central translucency following 3 days of culture. Addition of 1,000 epicardial cells at time 0 resulted in the most intact epicardial layer, showcased by fluorescence imaging. EPDCs epicardioids were smaller than cardioids (P ≤ 0.05), indicating that epicardial cells confers compactness. CD31 and MSLN staining showcased organized endothelial layering suggestive of coronary-like structures. H9C2 myoblasts viability was preserved in EPDC epicardioids as compared to MEC epicardioids (P ≤ 0.01). MEC epicardioids displayed 59% more endothelial cells compared to cardioids (P ≤ 0.001) and 49% more than EPDC epicardioids (P ≤ 0.01), suggesting a pro-angiogenic role of embryonic epicardial cells. Additionally, adult epicardial cells promoted healthy H9C2 morphology compared to Cardioids. Challenge with TGF-β1 resulted in increased collagen expression, a marker of fibroblast activation and enhanced cellular release, as confirmed by immunostaining and qPCR analysis. PH treatment determined a marked trend of increased nuclear area, indicative of hypertrophy.
DISCUSSION & CONCLUSIONS:
Epicardioids offer a novel platform to study adult epicardial communication with the main cell types of the heart. Recapitulating key aspects of epicardial interaction with myocardial and endothelial populations showcased by the structural organisation of endothelial structures and distinct morphological changes in H9C2 cells the presence of adult epicardial cells. This model enables functional assessment of epicardial influence on cardiac remodelling and may accelerate discovery in cardiac regeneration, drug screening, and disease modelling.