Background: Heart failure with preserved ejection fraction (HFpEF) remains a prevalent and growing condition, with limited therapeutic options. It is characterised by diastolic dysfunction and increased left ventricular stiffness resulting from excessive ECM remodelling. Cardiac fibroblasts (CFs) are central to HFpEF pathology, contributing to interstitial fibrosis and tissue stiffening, which impairs cardiac relaxation. Current rodent models of HFpEF fail to fully recapitulate the fibrotic phenotype observed in the clinics. In this project we leverage human induced pluripotent stem cells (hiPSCs), in accordance with the 3R principles (Replacement, Reduction, and Refinement), to model HFpEF in vitro and investigate fibrosis-related interactions within the broader cardiac microenvironment.
Purpose: This study aims to identify mechanisms underlying HFpEF-related fibrotic remodelling by developing a human-based in vitro model of HFpEF-associated fibrosis. We hypothesise that YAP (Yes-associated protein) signalling is driving fibrosis in HFpEF by activating fibroblasts to produce excess extracellular matrix (ECM) proteins.
Methods: We differentiated hiPSCs into cardiac fibroblasts (CFs) optimizing an established protocol for the epicardial lineage. We confirmed their identity by measuring the expression of fibroblast-specific markers with immunofluorescence and flow cytometry. To model the HFpEF-associated fibrotic phenotype, hiPSC-CFs were exposed to a cocktail of molecules recapitulating frequent comorbidity conditions of HFpEF, including hypertension, diabetes, obesity-related inflammation and lipotoxicity. Using advanced single-cell microscopy and image-based analysis, we quantified cell size, α-smooth muscle actin (αSMA) expression, nuclear translocation of YAP and ECM protein deposition, particularly fibronectin. Additionally, we performed RNA sequencing to assess transcriptional alterations indicative of fibrosis. This integrative approach enabled the systematic interrogation of HFpEF-associated pathways.
Results: Exposure to the HFpEF cocktail induced a marked increase in fibroblast size, consistent with hypertrophic remodelling. Image analysis revealed reorganization of αSMA fibres with reduced dispersion and greater alignment, forming contractile networks, a hallmark of fibroblast-to-myofibroblast transition. HFpEF treatment also altered subcellular localization of key profibrotic regulators. YAP, which was predominantly cytoplasmic under baseline conditions, shifted to the nucleus, indicating transcriptional activation. Importantly, treatment with verteporfin, a YAP inhibitor, mitigated these effects, restoring cell size, YAP localization, and fibronectin expression to levels comparable with controls.
Conclusion: We establish hiPSC-derived cardiac fibroblasts as a human model of HFpEF-associated fibrosis, recapitulating key cellular and molecular hallmarks of the disease. Verteporfin treatment restored cell morphology, YAP localization, and matrix remodelling to control-like states, providing a proof-of-concept for targeting YAP-dependent signalling. Together, these findings offer a platform for dissecting HFpEF pathophysiology and accelerating antifibrotic drug development.
