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. 2013 Dec 15;273(3):500-7.
doi: 10.1016/j.taap.2013.09.017. Epub 2013 Oct 1.

Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

Oksana Sirenko  1 , Evan F Cromwell, Carole Crittenden, Jessica A Wignall, Fred A Wright, Ivan Rusyn
Affiliations

Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

Oksana Sirenko et al. Toxicol Appl Pharmacol. .

Abstract

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30 min or 24 h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3 nM to 30 uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca(2+) flux readouts synchronous with beating, and cell viability. A number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration-response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-treated cells. BMC values were used for cardiotoxicity classification and ranking of compounds. Beat rate and several peak shape parameters were found to be good predictors, while cell viability had poor classification accuracy. In addition, we applied the Toxicological Prioritization Index (ToxPi) approach to integrate and display data across many collected parameters, to derive "cardiosafety" ranking of tested compounds. Multi-parameter screening of beating profiles allows for cardiotoxicity risk assessment and identification of specific patterns defining mechanism-specific effects. These data and analysis methods may be used widely for compound screening and early safety evaluation in drug development.

Keywords: AUC; BMC; Calcium flux; Cardiotoxicity; DMSO; EC(50); Phenotypic screening; Predictive toxicology; area under the curve; benchmark concentration; dimethyl sulfoxide; effective concentration at 50% of the maximum response; hERG; iPSC; induced pluripotent stem cell.

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Conflict of interest statement

Conflict of Interest:

O.S., E.F.C., and C.C. were employed by Molecular Devices, LLC, a company whose products and software were used to collect and analyze the data presented in this manuscript. J.A.W., F.A.W., and I.R. declare no conflicts of interest.

Figures

Figure 1
Figure 1
Concentration-dependent effects of screened compounds on peak width and beat rate of human iPSC-derived cardiomyocytes were visualized using hierarchical clustering and heatmaps. Colors represent changes in each parameter (see color scale bars). Values from duplicate plates were averaged to generate the heatmap. Compounds classified as cardiotoxic in Screen-Well™ Cardiotoxicity Library (107 chemicals) were clustered separately (top part of the figure) from those classified as non-toxic (24, bottom part of the figure). The complete list of compounds (131) used in this study is available as Supplemental Table 1.
Figure 2
Figure 2
Quantitative analysis of human iPSC-derived cardiomyocyte beating patterns (top left) was conducted using four-parameter logistic curve fits to the data. Before plotting, individual response values were normalized to plate-specific vehicle (DMSO) control values of each parameter. A benchmark response for the point-of-departure benchmark concentration in the concentration-response curve (vertical dashed lines) was defined as a one standard deviation departure from the mean for vehicle control wells (horizontal dashed lines).
Figure 3
Figure 3
Distributions of BMC values for non-toxic (white) and cardiotoxic (grey) compounds were plotted using box-and-whiskers plots (line - mean, box - inter-quartile range, whiskers - 5 and 95%, outliers – dots) for each parameter separately at 30 min (A) and 24 hr (B) time points. Differences in the means of BMCs between non-toxic and cardiotoxic compounds for each parameter were compared using t-tests and the level of significance is displayed with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; n.s., p>0.05).
Figure 4
Figure 4
Receiver Operating Characteristics curves that display the relationship between sensitivity and specificity were obtained from the analysis of the classification accuracy (cardiotoxic vs non-toxic labels of compounds in Screen-Well™ Cardiotoxicity Library) using BMC values (left – 30 min time point; right – 24 hr time point) for each of the parameters shown. AUC values for each ROC curve is displayed.
Figure 5
Figure 5
Principal component analysis (first two principal components shown with percent global variability explained indicated in parentheses) of chemical similarity vs. multi-parametric assessment of cardiotoxicity. A, Chemical diversity of Screen-Well™ Cardiotoxicity Library drugs using Dragon descriptors. B, Responses at each concentration for beat rate and peak shape (spacing, amplitude, rise, decay and width) for 30 min time point. C, Same as B for 24 hr time point. Non-toxic compounds are shown as blue circles. Cardiotoxic compounds are shown in red (triangles – compounds with known dopamine- and/or histamine-ergic activity, diamonds – compounds with known effects on ion channels, squares – other cardiotoxic compounds).
Figure 6
Figure 6
Relative cardiosafety ranking of Screen-Well™ Cardiotoxicity Library drugs. ToxPi scores (see Supplemental Tables 5 and 6) were used to create a relative ranking of screened drugs using multiparametric data collected from 30 min (A) and 24 hr (B) time points. Note that cell viability data for 30 min time point was collected 24 hrs after addition of chemicals to cells. Blue squares – non-toxic drugs, red squares – cardiotoxic drugs. Representative ToxPi-es for two non-toxic and two cardiotoxic compounds are shown to visualize the relative contribution of each parameter (represented in colors of each slice) to the overall score of a compound.
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