Stiffness-driven electrical remodeling: exploring mechanosensitive dynamics in hiPSC-CM

Abstract Background Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) present a valuable alternative to animal models for cardiovascular disease studies. Although hiPSC-CM display cardiogenic features, they lack the mature properties of adult cardiomyocytes. hiPSC-CM maturation is influenced by their microenvironment, particularly substrate stiffness, which impacts cellular remodeling and biomechanical properties. This study examined how biomechanical cues during culture affect hiPSC-CM maturation, focusing on cell geometry and substrate stiffness. We hypothesized that these factors influence excitation-contraction (EC) coupling and that different substrate stiffnesses activate mechanosensitive pathways, specifically the Yes-associated protein (YAP) signaling cascade, to modulate hiPSC-CM electrical and contractile functions. Purpose Our aim was to assess the effect of substrate stiffness on action potential (AP) parameters and EC-coupling, and to evaluate the mechanosensitive YAP dynamics and their long-term consequences on contractility in hiPSC-CM. Methods hiPSC-CM were cultured on stiff (glass, 25 GPa) and soft (PDMS, 28 kPa, 15 kPa) surfaces for up to 30 days. Cells were shaped into cuboid (adult CM-like) or hexagonal (control) forms by microcontact printing. 3D cultures were grown as spheroids and cuboid tissues in soft and stiff fibrin hydrogels. Structural remodeling of cardiac proteins and YAP translocation were assessed by immunocytochemistry and confocal imaging. APs, Ca2+ transients and contractions were measured optically. All analyses were performed using ImageJ and OriginPro software. Results Our data revealed strong myofibril alignment in cuboid cells and tissues, unlike hexagonally-shaped cells or spheroids. YAP nuclear translocation was significantly higher in stiff environments (p<0.0001). AP upstroke velocity was significantly faster, and AP duration prolonged on soft substrates (p<0.0001). Ca2+ transients on soft surfaces displayed faster release and decay rates, indicating functional maturation of Ca2+ handling. In addition, cell shortening increased on softer matrices, enhancing contraction efficiency. In hiPSC-CM fibrin hydrogels, full duration at half-maximum was shorter in soft compared to stiff hydrogels (FDHM: 358±9 ms and 550±18 ms, respectively, p<0.05), with significantly slower relaxation speed in stiffer hydrogels (stiff: 5.76±0.66 µm/s, soft: 8.50±1.23 µm/s, p<0.05). Conclusions These findings show that cell morphology and substrate stiffness influence the electro-mechanical response of hiPSC-CM, emphasizing the importance of mechanosensing in cellular adaptation to biomechanical load changes. These parameters are essential for further optimization and maturation of hiPSC-CM and their potential clinical use.