Professor University of Minnesota, Minnesota, United States
Introduction: : Ischemic heart disease remains a leading cause of death worldwide, partly due to the adult heart’s limited regenerative capacity. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer a promising platform for disease modeling and regenerative therapy. Despite this, clinical use is limited by a persistent immature phenotype in vitro. Epicardial-derived cells (EDCs) have emerged as key regulators of cardiomyocyte maturation through both paracrine and physical interactions. However, the molecular mechanisms underlying this process are not yet fully understood. Here, we integrate computational modeling and in vitro experiments to identify and validate GSK3 as a key modulator of cardiomyocyte functional maturation.
Materials and
Methods: : We developed a computational model of CM maturation that comprises 49 nodes and 60 reactions, utilizing logic-based differential equations3. This regulatory signaling network was informed by literature and preliminary experimental data. Sensitivity analysis of this model was performed by knocking down each node (individually) of the model and measuring it effect on all other nodes. To validate one of these predictions experimentally, we used a 2D co-culture system of hiPSC-CMs and EPDCs with or without TGF- inhibition to control epicardial epithelial-to-mesenchymal transition (EMT). Cultures were treated with GSK-3 inhibitor (CHIR) 24 hrs after cells were plated. Functional maturation was assessed via calcium transient analysis using Rhod-3 AM dye. Each condition was tested in two biological replicates (n=2) with six technical replicates.
Results, Conclusions, and Discussions:: Model analysis identified WNT and GSK-3 as top influences of cardiomyocyte maturation (Figure 1A). Validation of one of these findings showed that CHIR treatment enhanced calcium handling in both monocultures and co-cultures, consistent with computational predictions. In monocultures, CHIR increased the calcium transient amplitude and upstroke velocity, and reduced the time to peak, all of which are indicative of a shift toward a more mature phenotype (Figure 1 B-C). CHIR-treated co-cultures also demonstrated increased upstroke velocity and shortened time to peak compared to untreated co-cultures, though changes in amplitude were more modest (Figure 1 B-C). These findings suggest that GSK-3 inhibition promotes CM functional maturation and that this effect is preserved and even modulated in the presence of epicardial cells. Computational modeling predicted, and experimental results confirmed that GSK-3 inhibition enhances functional maturation of hiPSC-derived cardiomyocytes. These findings highlight the value of integrating computational predictions with in vitro validation to identify molecular targets that can advance cardiac tissue engineering systemically. Future work will refine the model to capture more complex dynamics and extend validation to 3D culture systems.
