Obviously T changes are not

Obviously, T2 changes are not specific for certain alterations in ABT-199 current, as modulators with different modes of action (ATX-II, Bay K-8644, E-4031) all result in similar changes. Moreover, relaxation time was also prolonged by EMD-57033, a relatively pure calcium sensitizer that prolongs relaxation by its direct effect on the myofilaments, and relaxation time was shortened by the cAMP-dependent drugs isoprenaline and rolipram, acting via phosphorylation of PLN and several myofilament proteins (Figure 5). Interestingly, neither verapamil nor TTX affected relaxation time. Their effect was visible as a concentration-dependent decrease in force and rate, respectively. The lack of verapamil effects on T2 is important for two reasons. (1) It is consistent with the effect of varying extracellular Ca2+ concentrations, strongly affecting peak force but not contraction kinetics. (2) Verapamil blocks IKr in addition to ICa,L and is a classic example of a drug that would have failed in hERG assays, but has never been associated with proarrhythmic effects or QT prolongation. The lack of a T2 signal in hiPSC-EHTs is in line with clinical safety. Thus, the hiPSC-EHT system offers a high-content readout of contractile function, which integrates effects on depolarizing and repolarizing ion currents, myofilaments, and SR-based Ca2+ cycling without directly indicating the respective mechanism of action. It seems, therefore, that this system is well suited for an initial screening of cardiac effects of drugs. The ability to detect the effects of ion-channel modulators was further challenged with a set of (previously) approved drugs, classified for their different potential to induce torsades-de-pointes arrhythmia (Redfern et al., 2003). The correlation depicted in Figure 7 demonstrates that prolongation of T2 in hiPSC-EHTs is a sensitive surrogate for APD90 prolongation. Given the key role of APD prolongation in drug-induced cardiac proarrhythmia (Fermini et al., 2015), a surrogate for APD prolongation might address an important aspect of in vitro cardiac safety assessment. This conclusion is strengthened by the lack of T2 effects of four compounds known to be safe (aspirin, paracetamol, ampicillin, and verapamil).
Experimental Procedures
Author Contributions
Acknowledgments We thank Lisa Krämer, Giulia Mearini, June Uebeler, Maksymilian Prondzynski, Sari Panjaitan, Bärbel Ulmer, Umber Saleem, Sandra Laufer, and Aya Shibamiya (HEXT Stem Cell Core Facility, UKE Hamburg) for their support. We greatly appreciate the assistance of Kristin Hartmann and Susanne Krasemann (HEXT Mouse Pathology Core Facility, UKE Hamburg), in processing histological and TEM samples. This study was supported by Deutsche Forschungsgemeinschaft (DFG Es 88/12-1, DFG Ha 3/1), the British National Centre for the Replacement Refinement & Reduction of Animals in Research (NC3Rs CRACK-IT grant 35911-259146), the European Research Council (ERC-AG IndivuHeart), the EU (FP7 Biodesign), the German Centre for Cardiovascular Research (DZHK), and the German Ministry of Education and Research (BMBF), British Heart FoundationRM/13/30157, the German Heart Foundation, the Freie und Hansestadt Hamburg, and Era-Net E-RARE (01GM1305). I.M., A.E., M.H., T.E., and A.H. are co-founders of EHT Technologies GmbH, Hamburg.
Introduction Overexpression of four transcription factors (OCT4, SOX2, KLF4, and MYC) reprograms differentiated cells to become induced pluripotent stem cells (iPSCs). The global epigenomic changes that accompany reprogramming include histone modification, DNA methylation, expression of non-coding RNAs, and reactivation of the inactive X chromosome (Kim et al., 2014; Papp and Plath, 2013). iPSCs maintain the genetic composition of donor cells, and thus have been proposed to model human diseases in vitro through differentiation into target cell types. In addition, iPSCs can provide autologous cells for cell replacement therapy (Wu and Hochedlinger, 2011). However, studies have shown that iPSCs contain localized aberrant epigenetic states compared with human embryonic stem cells (hESCs) despite their high similarity (Bock et al., 2011; Lister et al., 2011). Understanding the reprogramming mechanisms and developing novel reprogramming technologies to minimize the abnormality of iPSCs are critical for the future use of iPSCs.