Mitochondria Clearance Enables Macrophage-Driven Maturation of iPSC-Derived Cardiomyocyte Metabolism.

Background: Immaturity of stem cell-derive cardiomyocytes limits their use in tissue engineering applications. Macrophage contributions to the development of cardiomyocytes have not yet been fully established. While some recent studies have added macrophages to stem cell-derived models of the human myocardium, these previous approaches do not replicate the early colonization of the heart. Due to their importance in regulating cardiomyocyte metabolism, we hypothesized that developmentally informed addition of macrophages to cardiomyocytes would improve cardiomyocyte maturity. Methods: We generated cardiomyocytes and embryonic-like macrophages from a single cell line. Macrophages were added to developing cardiomyocytes 8, 16, and 19 days after induction of differentiation, based on changes in cardiomyocyte media formulation at these stages. Cardiomyocytes were cultured until 30 days post differentiation, where they were prepared for analysis. Metabolism was measured through Seahorse Mitochondria Stress assays. Corresponding changes in subcellular structures were measured through high-resolution microscopy. Mitophagy in cardiomyocytes was measured through the fluorescent reporter mtKeima. Results: Addition of macrophages to cardiomyocytes 8 days after the induction of differentiation results in a significant increase in cardiomyocyte basal and maximal metabolism. Developing cardiomyocytes shed lowly polarized mitochondria, which are taken up by macrophages. As a result, cardiomyocytes adopt an adjusted mitochondria network architecture featuring less interconnected mitochondria. Mitophagy flux measurements show that cardiomyocytes develop more active mitophagy programs while in coculture with macrophages. Pharmacological inhibition of mitophagy reveals that this interaction is dependent on macrophage MerTK-mediated reception of cardiomyocyte-derived mitochondria material. Conclusions: These results improve our understanding of the responsibility of macrophages in the development of cardiomyocyte metabolism. We establish interactions between macrophages and developing cardiomyocytes as essential to produce more mature cardiomyocytes and physiologically relevant models of the human myocardium. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-026-00915-z.