Force generation of cardiomyocytes in engineered environments

Cardiomyocyte contraction is essential for the pumping action of the heart and deteriorates after myocardial damage, either as a consequence of irreversible cardiomyocyte injury or stiffening of the extracellular matrix, a process described as fibrosis. Cell geometry and substrate stiffness not only influence sarcomere architecture and contractility, they also determine how much work the cardiomyocytes can transfer to their environment. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes provide a model system to examine these effects in a controlled manner, enabling us to mimic key features of the cardiac environment in vitro. Here we show that geometric confinement and substrate stiffness influence different aspects of cardiomyocyte force generation. By combining traction force microscopy and live-cell imaging of hiPSC-derived cardiomyocytes on soft and patterned substrates, we find that geometric confinement leads to longer resting sarcomeres and more pronounced sarcomere shortening on substrates with both physiological (10 kPa) and fibrotic (30 kPa) stiffness. Interestingly, the substrate stiffness itself has little effect on resting sarcomere length, but influences the peak contractile stress. On 10 kPa substrates, confined cells generate higher peak contractile stresses than unconfined cells, whereas this difference is not observed at 30 kPa. Confinement also results in faster mechanical relaxation at 10 kPa, with no detectable difference at 30 kPa. The combination of defined cell geometry and physiological stiffness leads to longer resting sarcomere lengths, more pronounced sarcomere shortening, higher peak contractile stress, and faster relaxation, features associated with a more mature cardiomyocyte phenotype. These findings show that the mechanical consequences of cell geometry depend on substrate stiffness and, therefore, both aspects have to be considered when employing hiPSC-derived cardiomyocytes as a model system in mechanobiology research, disease modeling and drug testing.