br Results br Discussion We demonstrated a significant posit

Results
Discussion We demonstrated a significant positive correlation between the susceptibility of hiPSC-CMs to Mox-induced FPD prolongation and QT prolongation in healthy individuals within a certain concentration range. Thus, in vitro assays using hiPSCs derived from healthy individuals may improve cardiotoxicity risk assessment in drug development. The combination with assay models to assess genetic susceptibility (Liang et al., 2013) would further enhance the prediction of SAE occurring in small subgroups. Despite the small study cohort (n = 10), the data were considered reliable because the correlations in individuals were highly consistent across different parameters and all measurements were obtained in a blinded manner. It will be necessary to evaluate the practical usability of this system in a large-scale clinical study including a large number of volunteers. An MEA was used to investigate the relationship between in vivo electrocardiography (ECG) data and in vitro data in iPSC-CMs because the synchronous beating of a cultured cardiac myocyte monolayer in vitro shows electric patterns similar to those of the heart. Moreover, MEAs may be most useful when used not only to evaluate the electrophysiological properties of cardiomyocytes but also to correlate them with tissue networks (Sallam et al., 2015). However, one major concern related to MEA is that the data depend on the filtering of the signal from cells, and FPD cannot be determined in case of absent or bimodal field potential peaks. To address these concerns, we only measured FPDs including high peaks. Thus, we considered the use of an MEA for a cell sheet appropriate for comparison with ECG QT intervals. The slope values of Mox-induced FPD prolongation were affected by Mox concentration, and significant positive correlations were observed at Mox concentrations of 0–10 μM. Similar results were observed in comparison with QT prolongation values at Cmax. The maximum Mox insulin receptor concentration was 3.01–5.35 mg/L (6–11 μM) (Table S1), and Mox is known to have a low protein-binding rate (approximately 40%) (Stass and Kubitza, 1999). In addition, it has been reported that electrophysiological properties and drug responses of human embryonic stem cell-derived cardiomyocytes match clinical observations on QT prolongation at similar concentrations (Braam et al., 2010), suggesting that the concentrations that yielded significant positive correlations in vitro were in the range of plasma exposure in the clinical study. The non-significant correlation at concentrations >30 μM in vitro might be explained by off-target effects at supra-pharmacological concentrations. We observed a significant positive correlation at Mox concentrations of 0–3 μM for ΔQTcF-Cmax and ΔΔQTcF-Cmax, but not for ΔQTcF-slope and ΔΔQTcF-slope; one of the reasons may be the different algorithm used to quantify susceptibility in vivo. ΔQTcF- and ΔΔQTcF-slope values were affected by hysteresis, which is a delay in equilibration between plasma concentrations and QT changes. In fact, the range for QT prolongation was larger at later than at earlier time points at the same plasma Mox concentration in some individuals, indicating counterclockwise hysteresis (Figure S2B). In addition, we observed variability in the data at 0–3 μM (Figure 3E). For a more accurate correlation it will be necessary to reduce the noise in the in vitro measurement. We could not investigate detailed cellular mechanisms to explain the correlation between in vivo and in vitro data because of unavailability of heart tissue samples from each participant; therefore, we investigated the possible role of genetic variation. hiPSC-CMs with specific mutations have phenotypes that are similar to the disease phenotype, including long QT syndrome (Liang et al., 2013; Itzhaki et al., 2011). However, the participants had no genetic mutations that would result in amino acid changes in target-binding sites for Mox or other drugs associated with the induction of QT prolongation. Moreover, the baseline QT ranges were normal (410 ± 14 ms) (Burke et al., 1997) in the study participants. Although some genes such as KCNE4 and KCNH2 were differentially expressed among hiPSC-CM lines, these differences did not correlate with in vivo data. In SNP analysis only one polymorphism, (rs81204) in KCNQ1, was present in two volunteers highly susceptible to Mox. On the other hand, we could not confirm a correlation with a polymorphism in FRMD6 reported in a GWAS (Behr et al., 2013) in our volunteers. Interestingly, five SNPs in CACNA1C related to cLQTS8 and three SNPs in RYR2 related to drug-induced QT prolongation (Kääb et al., 2012; Ramirez et al., 2013) were different between volunteers with low and high susceptibility to Mox. On the other hand, three SNPs in PALLD related to antipsychotic drug-induced QT prolongation (Aberg et al., 2012) showed differences between volunteers with low and high susceptibility. This might be explained by the fact that these are different types of drugs. Interestingly, the frequency of these three SNPs differs between Japanese (MAF 0.1–0.2) and Europeans (MAF 0.4–0.5). Although the relationship between this polymorphism and phenotypes was difficult to determine because of the small cohort, these SNPs might be one of the candidates to indicate evidence in the relationship of different susceptibility between in vivo and hiPSC-CMs. Further GWASs using a larger number of participants with diverse polymorphisms are warranted. The hiPSC-CM model has some limitations (Denning et al., 2016), such as the immature phenotype (Zhu et al., 2014) or indeterminate subtypes of cardiomyocytes (David and Franz, 2012). However, they are useful for high-throughput plate assays and high-content imaging (Mercola et al., 2013) because there is no contamination with other cell types (Burridge et al., 2016). In addition, extension of the culture period induces maturation in hiPSC-CMs (Yang et al., 2014). Therefore, the immature features and gap in differentiation periods among hiPSC-CMs derived from an individual might limit the recapitulation of drug susceptibility to Mox between a subject and hiPSC-CMs derived from the subject. To obtain a high correlation score between human susceptibility and hiPSC-CMs, it is necessary to improve the differentiation method to prepare more mature cardiomyocytes and control their different maturation states.