From cells to structure: the endothelial contribution to extracellular matrix architecture in dilated cardiomyopathy

Abstract Dilated cardiomyopathy (DCM) represents the third leading cause of heart failure worldwide. LMNA mutations have been demonstrated to induce structural and functional myocardial abnormalities. Disease progression has been strongly associated with endothelial cell (EC) dysfunction, which drives extracellular matrix (ECM) remodelling and fibrosis. Ex vivo analyses of DCM myocardium have consistently demonstrated elevated ECM accumulation and capillary rarefaction. In order to examine these vascular alterations in vitro, two- and three-dimensional (2D and 3D) endothelial models were established using human induced pluripotent stem cell-derived ECs (hiPSC-ECs) and patient-derived primary ECs (pECs), enabling the combined use of both cell sources and the assessment of their cell type-specific ECM production. This study aims to clarify the vascular contribution to DCM pathophysiology using a patient-specific 3D in vitro model with a dedicated focus on cell-derived ECM deposition. Differences in ECM production between 2D and 3D cultures of hiPSC-ECs and pECs were examined, allowing direct comparison of their matrix remodelling behaviours under DCM-associated conditions. This approach supports translational research, mid-throughput organoid generation, and "clinical trials in a dish" applications for disease-specific drug screening and therapeutic testing. Microvascular pECs were isolated from explanted cardiac tissue of patients with end-stage heart failure. Ex vivo myocardial samples were analysed using immunohistochemistry, whereas 2D and 3D cultures of hiPSC-ECs and pECs were assessed by immunocytochemistry. Quantification of confocal and fluorescence microscopy images was performed in ImageJ using integrated density normalised to area. Spheroids were generated through cell aggregation, subsequent embedding in ECM, and cultivation in low-attachment U-bottom plates to yield organoids. Cellular composition and cell-derived ECM deposition were systematically evaluated to determine the contributions of each EC source to matrix remodelling. Cardiac tissues from LMNA-mutated DCM patients (NYHA class IV, LVEF 25% 15–40, n = 10) were compared with healthy controls (n = 7). DCM tissues and LMNA-mutant EC cultures exhibited significantly increased ECM deposition relative to controls. Notably, hiPSC-ECs and pECs demonstrated distinct ECM production profiles, indicating cell-type-specific matrix remodelling responses. These disparities highlight the critical impact of EC origin when modelling DCM-associated ECM dysregulation. The 3D endothelial model established in this study was robustly reproduced across both hiPSC-ECs and pECs. Owing to its ability to recapitulate vascular pathologies associated with DCM, particularly enhanced cell-derived ECM deposition, this platform represents a powerful tool for studying disease mechanisms and evaluating therapeutic strategies in vitro.