Preclinical drug safety screening using induced pluripotent stem cell-derived cardiomyocytes in engineered heart tissue format

Abstract

Cardiovascular side effects are an important major cause for attrition during the process of drug development. Preclinical cardiac assessment relies on in vitro and in vivo animal experimentation. The predictive value of these experiments is low. The principally unlimited availability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) was the basis for the development of assays for pre-clinical cardiac assessment based on this cell source. As one example, culturing hiPSC-CM in three-dimensional fibrin based engineered heart tissues (EHT) and semi-automated video optical analysis of contractility was developed at the Department of Experimental Pharmacology and Toxicology, University Medical Centre Hamburg-Eppendorf. This provides an innovative test-bed for cardiac assessment. The present thesis aimed to combine the established contractility test system with the analysis of calcium transients (CaT) by using genetically encoded calcium indicators (GECIs). The suitability of the test system was evaluated in the context of an international, multi-site blinded screening project. Viral transduction of EHTs to express GECIs (GCaMP5G and GCaMP6f) was established with lentivirus and adeno-associated virus 6 (AAV6). The video-optical EHT contractility test system was expanded to sequentially analyse GCaMP6f fluorescence intensity as a surrogate for CaT. Both GECIs showed specific signals with high signal to noise ratios. EHTs transduced with GCaMP5G showed slower on- and off-kinetics. The test system was validated with a set of indicator compounds (e.g. isoprenaline, nifedipine, BayK-8644 and digoxin), which revealed compound specific effects on force and CaT, well compatible with the respective mechanisms of action. The advanced test system was used to study force- and CaT-frequency relationship in the presence of ivabradine (300 nM) which reduces baseline frequency. Under these conditions, a positive force- and CaT-frequency relationship was demonstrated in a range of 0.5-1.5 Hz but not at higher frequencies. Omecamtiv mecarbil was also analysed in the presence of ivabradine and showed a pronounced positive inotropic effect at low frequencies and a small positive inotropic effect at higher frequencies (+240% at 1.0 Hz, +63% at 2.5 Hz, reverse frequency dependency). The test system was subjected to a blinded screening of 27 compounds with half-log concentration response curves. Most of the compounds showed the expected effects on inotropy with a predictive accuracy of 75% for positive, 88% for negative inotropes, and 77% for neutral inotropes. Small effect size of positive inotropic effects, delayed cardiotoxic negative inotropic effects, and misclassification of neutral inotropes because of normalization to pooled time controls were identified as relevant weaknesses of this screening approach. In a separate part of this thesis, the regulation of force by different isoforms of phosphodiesterases (PDE) in hiPSC-CM EHTs was investigated that revealed a predominance of PDE4 over PDE3 unlike adult human heart where PDE3 is the most important isoform. PDE4 dominance is likely an indicator of the immaturity of hiPSC-CM. Overall, this study described the combination of CaT analysis with the video-optical contractility analysis in hiPSC-CM EHTs as a meaningful extension and the results emphasis the potential use of this model for preclinical cardiac drug assessment applications