Background: To get insight into the mechanism of action of different compounds affecting cardiac contraction and relaxation, we aimed to established an in-vitro system for dual measurements of contraction force and calcium transient (CaT) in human engineered heart tissues (EHT) using genetically encoded calcium indicators (GECIs).
Methods: Three-dimensional, force-generating fibrin-based EHTs were prepared from hiPSC cardiomyocytes and transduced with GECIs (GCaMP5G or GCaMP6f). We sequentially (10 sec delay) measured contraction force (video-optical recording) and CaT (fluorescent light intensity) and analysed 8 compounds affecting cardiac contraction and relaxation: myofilament Ca2+ sensitizer (EMD-57033), L-type calcium channel agonist/antagonist (Bay K-8644, nifedipine), hERG blocker (E4031), ryanodine receptor antagonist (ryanodine), beta-adrenergic agonist (isoprenaline), SR Ca2+-ATPase inhibitor (thapsigargin), Na+/K+ ATPase inhibitor (digoxin).
Results: Both GECIs showed a strong signal to noise ratio with example values of 20,000 in 1 mM Ca2+. GCaMP6f showed faster on and off kinetics than GCaMP5G, better reflecting physiological CaT kinetics. EMD-57033 (10 μM) increased force (+170%) without change in CaT. Bay K8644 (300 nM) increased force amplitude (+55%), relaxation time (+64%) and calcium decay time (+55%). Nifedipine (100 nM) reduced force (-79%), contraction time (-30%), CaT (-76%) and calcium rise time (-26%). E4031 (300 nM) prolonged relaxation (+26) and calcium decay time (+19). Ryanodine (10 μM) decreased force (-37%) and CaT (-49%). Isoprenaline (10 nM) led to a positive inotropic (+32%), lusitropic (-17%) effect and increased CaT (+13%) and decreased calcium decay time (-21%). Thapsigargin (3 μM) decreased force (-28%) and CaT (-33%). Digoxin (0.3 μM) increased force amplitude (+36%) and CaT (+46%), reduced contraction time (-14), relaxation time (-18%), calcium rise (-18%) and calcium decay time (-23%). Force/calcium loops also revealed compound-specific changes illustrating the mechanism of action.
Conclusion: Dual Ca2+ and force measurements in hiPSC-EHTs provide important information on drug actions on cardiac excitation contraction coupling under stable experimental conditions.