Blinded, Multicenter Evaluation of Drug-induced Changes in Contractility Using Human-induced Pluripotent Stem Cell-derived Cardiomyocytes

Abstract

Animal models are 78% accurate in determining whether drugs will alter contractility of the human heart. To evaluate the suitability of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for predictive safety pharmacology, we quantified changes in contractility, voltage, and/or Ca2+ handling in 2D monolayers or 3D engineered heart tissues (EHTs). Protocols were unified via a drug training set, allowing subsequent blinded multicenter evaluation of drugs with known positive, negative, or neutral inotropic effects. Accuracy ranged from 44% to 85% across the platform-cell configurations, indicating the need to refine test conditions. This was achieved by adopting approaches to reduce signal-to-noise ratio, reduce spontaneous beat rate to ≤ 1 Hz or enable chronic testing, improving accuracy to 85% for monolayers and 93% for EHTs. Contraction amplitude was a good predictor of negative inotropes across all the platform-cell configurations and of positive inotropes in the 3D EHTs. Although contraction- and relaxation-time provided confirmatory readouts forpositive inotropes in 3D EHTs, these parameters typically served as the primary source of predictivity in 2D. The reliance of these "secondary" parameters to inotropy in the 2D systems was not automatically intuitive and may be a quirk of hiPSC-CMs, hence require adaptations in interpreting the data from this model system. Of the platform-cell configurations, responses in EHTs aligned most closely to the free therapeutic plasma concentration. This study adds to the notion that hiPSC-CMs could add value to drug safety evaluation.

Keywords: CRACK-IT project; alternatives to animal testing; cardiomyocytes; contractility; electrophysiology; human-induced pluripotent stem cells; inotropy; predictive toxicology; safety pharmacology.

Figure 1.

A, Contraction analysis across the platform-cell combinations for PIs. Examples are given to illustrate when some or all platform-cell combinations enable correct or incorrect predictions of the PIs, epinephrine, and levosimendan, respectively. The table indicates where decision making was guided by data from Ca²⁺ transients and/or voltage, with example data provided for epinephrine and verapamil in Supplementary Figures 4A and 4B. Red dotted line is free therapeutic plasma concentration (FTPC). Dunnett’s stats versus vehicle control: *p < .05; **p < .01; ***p < .001; ****p < .0001. B, Contraction analysis across the platform-cell combinations for NIs. Examples are given to illustrate when some or all platform-cell combinations enable correct or incorrect predictions of the NIs, verapamil, and sunitinib, respectively. The table indicates where decision making was guided by data from Ca²⁺ transients and/or voltage, with example data provided for epinephrine and verapamil in Supplementary Figures 4A and 4B. Red dotted line is FTPC. Dunnett’s versus vehicle control: *p < .05; **p < .01; ***p < .001; ****p < .0001. C, Contraction analysis across the platform-cell combinations for NEs. Examples are given to illustrate when some or all platform-cell combinations enable correct or incorrect predictions of the NEs, acetylsalicylic acid, and captopril, respectively. Red dotted line is FTPC. Dunnett’s stats versus vehicle control: *p < .05; **p < .01; ***p < .001; ****p < .0001. Abbreviations: CO, CellOPTIQ; EHT, engineered heart tissue; TTM, Triple Transient Measurement.

Figure 2.

A, Sensitivity of platform-cell combinations for predicting positive inotrope (PIs). Where PIs were correctly predicted, data are presented as percent change relative to baseline for any of the 3 contractility parameters (CA, contraction amplitude; CT, contraction time; RT, relaxation time). The bolded black text indicates where significance was reached using Dunnett’s stats versus vehicle control and p < .05. B, Sensitivity of platforms-cell combinations for predicting negative inotropes (NIs). Where NIs were correctly predicted, data are presented as percent change relative to baseline for any of the 3 contractility parameters (CA, contraction amplitude; CT, contraction time; RT, relaxation time). The bolded black text indicates where significance was reached using Dunnett’s stats versus vehicle control and p < .05. Abbreviations: CO, CellOPTIQ; EHT, engineered heart tissue; FPTC, free plasma therapeutic concentration; NR, not recorded; TTM, Triple Transient Measurement.

Figure 3.

Most informative contractility marker. Parameters that led to the correct prediction of drug responses were assessed. Data show the breakdown by percentage for the parameters (CA, contraction amplitude; CT, contraction time; RT, relaxation time) that reached significance at the lowest concentration (Dunnett’s stats vs vehicle control and p < .05). White boxes (NS, not significant) indicate where predictions were made due to a trend rather than reaching significance, and/or by guidance from Ca²⁺ and/or voltage data. Abbreviations: CO, CellOPTIQ; EHT, engineered heart tissue; TTM, Triple Transient Measurement.

Figure 4.

Refined culture conditions increase predictivity of positive inotropes (PIs) in 2D monolayers of hiPSC-CMs. Contraction analysis was carried out on the 8 PIs from the drug test set using the CO:R-PAT platform-cell combination. Only CT and RT are shown because data in Figure 3 showed these to be the most informative parameters for PIs. Whereas testing in serum-/protein-free medium failed to identify any PIs correctly (A, web tool), the slowed beat rate and improved signal-to-noise ratio afforded by culture in RPMI-B27 allowed correct identification of 6/8 PIs by significant decreases in CT and/or RT. Red dotted line is free therapeutic plasma concentration. Dunnett’s stats versus vehicle control: *p < .05; **p < .01; ***p < .001; ****p < .0001.

Figure 5.

Slowed beat rate increases the predictivity of positive inotropes (PIs) in 3D engineered heart tissues (EHTs). Contraction analysis was carried out using EHTs with 6 of the PIs from the drug test set: epinephrine (Epi), forskolin (For), levosimendan (Lev), pimobendan (Pim), dobutamine (Dob), and milrinone (Mil) which had been predicted with variable accuracy (Figure 4A, web tool). In all cases, the drugs (applied as a high concentration bolus) increased contraction amplitude (CA) but only at 0.5 and 0.7 Hz, and sometimes at 1 Hz, with the largest effects seen in contraction time (CT) also often occurring at these frequencies. Scatter plots show percentage changes relative to baseline at respective frequency. Averaged peaks for force are shown for baseline (BL, black peaks) versus after treatment in EHTs paced at 0.5 Hz (blue peaks) or 2.0 Hz (red peaks). Dunnett’s stats versus baseline at respective frequency: *p < .05; **p < .01; ***p < .001; ****p < .0001.