Cardiac Work Inhibits Cell Cycle Activity in Engineered Heart Tissue

Background: Cardiomyocytes exit the cell cycle postnatally and the adult heart possesses only a very limited regenerative capacity. The question of why cardiomyocytes lose their regenerative potential is unanswered. One explanation could be the continuous need for work that precludes cell division. We generated human pluripotent stem cell-derived cardiomyocytes (CM), in which contractility could be inhibited reversible with small molecules, to assess this hypothesis experimentally.

Methods: A pharmacological selective actuator module (PSAM), consisting of a modified nicotinergic acetylcholine receptor and the ion pore domain of the glycine receptor (PSAM-GlyR) was integrated in the AAVS1-locus of induced pluripotent stem cells (iPSC). PSAM-GlyR iPSC were differentiated to CM. CM were cast into three-dimensional engineered heart tissue (EHT). Force and frequency were measured by video-optical analysis. Troponin I concentration was measured in the supernatant to assess sarcomere turnover. EHTs were harvested on day 21 and 49 of culture and processed for histology. Cell cycle activity was assessed histologically by Ki67 staining.

Results: PSAM-GlyR EHT started to beat after 6 days in culture. In the intervention groups PSEM89S was applied from the beginning of culture either for 21 or for 49 days. Activation with the small molecular pharmacologically selective effector molecule 89S (PSEM89S) resulted in a depolarization block and PSA-GlyR EHT in the intervention groups did not start to beat. Yet, all EHT developed morphologically similar irrespective of beating. Troponin I concentration was lower in the medium of EHT that did not beat (day 21: 7.8 ± 0.5 pg/mL vs. 119.3 ± 17.0 pg/mL, day 49: 15.3± 0.9 pg/mL vs. 297.2 ± 203.3 pg/mL). In accordance sarcomere organization was lower in these EHT. After PSEM89S wash-out EHTs started to beat within 120 minutes on day 21 or 49 respectively. After 7 days they developed similar forces as the time-matched controls. Troponin concentration increased in the media after the EHT started to beat (99.9 ± 36.6 pg/mL). There was no difference in Ki67 positive nuclei at day 21 (beating control: 10.2 ± 0.5% vs. stopped: 13.8 ± 3.5%). However, cell cycle activity decreased in the beating controls over time (6.3 ± 1.9% at day 49). In contrast it remained higher in the PSAM-GlyR EHT that did not beat (12.1 ± 1.4%).

Conclusion: Inhibition of cardiac contractility prevented sarcomere assembly and resulted in higher cell cycle activity, providing first evidence that cardiac work inhibits cell cycle activity.