Incomplete maturation is a major limitation of pluripotent stem cell-derived cardiomyocyte models. Cultivation in a three-dimensional environment is one way to (partially) overcome this limitation, indicating that cardiac work stimulates cardiomyocyte (CM) maturation. We have recently developed engineered heart tissue (EHT) models based on human induced pluripotent stem cell derived CMs (hiPSC-CMs) in which contraction can be switched off reversibly (off-on switch). In the present study we used EHTs with a chemogenetic off-on switch to dissect out the influence of cardiac work on tissue maturation.

CRIPSR/Cas9 was used to knock-in a genetically engineered ligand-gated anion channel consisting of the ligand-binding domain of the nicotinic acetylcholine receptor and the ion pore domain of the glycine receptor (pharmacologically selective actuator module; PSAM-GlyR) into human induced pluripotent stem cells (hiPSC). PSAM GlyR-hiPSCs were subjected to cardiac differentiation. CMs were used for the generation of three-dimensional fibrin-based EHTs. Video-optical analysis and sharp microelectrode measurements were performed for the physiological characterization of PSAM GlyR-EHTs. Application of the small molecular pharmacological selective effector molecule 89S (PSEM^89S) was used to silence PSAM GlyR EHTs.  PSAM GlyR EHTs were treated for one, seven or twenty-one days either from the beginning of the culture period or after a run-in cultivation time of 21 days. EHTs were then cultivated for an additional seven days after PSEM^89S washout. Video-optical analysis were performed regularly during the cultivation time. EHTs were harvested for histological analysis at several, pre-defined time points.

PSAM GlyR-EHTs developed similar to control EHTs and showed a steady increase in contractility over the first weeks in culture. Treatment with the PSAM-specific agonist PSEM^89S resulted in a depolarization block and reversibly stopped EHT contractility. EHTs treated with PSEM^89S from the beginning of the culture period did not start to contract but immediately started to beat coherently after PSEM^89S washout and reached similar forces as the respective time-matched controls, indicating functional maturation in the absence of contractility. PSAM-GlyR EHTs that were silenced after an initial maturation period of 21 days similarly stopped to beat upon PSEM^89S application. After PSEM^89S wash-out they re-started to beat and did not only reach contractility prior to silencing but reached the higher contractility of the time-matched controls within 7 days. Alpha actinin staining displayed that the sarcomere structure in EHTs disassembled up on contractility off-switch but reassembled within seven days showing a similar pattern as in control EHTs.

 

The observation that EHTs which did not work for prolonged periods of time reached similar forces as their respective time-matched controls within a short time after initiation of contractility indicates cardiomyocyte development is partially independent from mechanical work. Histological analysis however showed that mechanical work is indeed a critical factor for the development cardiomyocyte structure. Finally, our approach provides a versatile tool for future studies in different fields such as cardiac cell biology, regenerative medicine and disease modeling.