Mechanosensation mediates volume adaptation of cardiac cells and spheroids in 3D

With the adoption of 3-dimensional (3D) cell culture for in vitro modelling of cardiac function and regenerative medicine applications, there is an increased need to understand cardiomyocyte mechanosensation in 3D. With existing studies of cardiomyocyte mechanosensation primarily focussed on the behaviour of individual cells in a 2-Dimensional context, it is unclear whether mechanosensation is the same in a 3D, multicellular context. In this study, H9C2 cardiac-derived myoblasts were encapsulated as individual cells and as cell spheroids within stiffness gradient gelatin methacryloyl (GelMA) hydrogels to investigate individual and collective cardiac cell mechanosensation in 3D. Over a 3.68–17.52 ​kPa stiffness range, it was found that H9C2 cells have a limited capacity to adapt their volume to increasing substrate stiffness, demonstrated by the lack of changes in cell volume and shape across the stiffness gradient. Morphological trends were reflected by the expression of the mechanomarkers YAP, MRTF-A and Lamin-A, which were better correlated with cell and nuclear volume than with substrate stiffness. The localisation of YAP and MRTF-A were dependent on the relative volumes of the cytoplasm and nucleus while Lamin-A expression was elevated with increasing cytoplasmic and nuclear volumes. When cultured as spheroids rather than as individual cells, H9C2 cells adopted a distinct morphology with comparably smaller nuclei than individually cultured cells, while retaining the same overall cell volume. As spheroids, H9C2 cells were sensitive to stiffness cues, shown by decreasing YAP and MRTF-A nuclear localisation, increasing Lamin-A expression, and increasing vinculin expression with increasing substrate stiffness. Like the individually cultured H9C2 cells, mechanomarker expression was correlated to volume adaptation. With increasing cytoplasmic volume, YAP and MRTF-A became less nuclear localised, vinculin expression was increased, and with increasing nuclear volume, the Lamin-A expression fincreased. Together, these data suggest that cardiac cell volume adaptation may be enhanced by cell-cell interactions.