Bayesian approach enabled objective comparison of multiple human iPSC-derived Cardiomyocytes' Proarrhythmia sensitivities.

The one-size-fits-all approach has been the mainstream in medicine, and the well-defined standards support the development of safe and effective therapies for many years. Advancing technologies, however, enabled precision medicine to treat a targeted patient population (e.g., HER2+ cancer). In safety pharmacology, computational population modeling has been successfully applied in virtual clinical trials to predict drug-induced proarrhythmia risks against a wide range of pseudo cohorts. In the meantime, population modeling in safety pharmacology experiments has been challenging. Here, we used five commercially available human iPSC-derived cardiomyocytes growing in 384-well plates and analyzed the effects of ten potential proarrhythmic compounds with four concentrations on their calcium transients (CaTs). All the cell lines exhibited an expected elongation or shortening of calcium transient duration with various degrees. Depending on compounds inhibiting several ion channels, such as hERG, peak and late sodium and L-type calcium or IKs channels, some of the cell lines exhibited irregular, discontinuous beating that was not predicted by computational simulations. To analyze the shapes of CaTs and irregularities of beat patterns comprehensively, we defined six parameters to characterize compound-induced CaT waveform changes, successfully visualizing the similarities and differences in compound-induced proarrhythmic sensitivities of different cell lines. We applied Bayesian statistics to predict sample populations based on experimental data to overcome the limited number of experimental replicates in high-throughput assays. This process facilitated the principal component analysis to classify compound-induced sensitivities of cell lines objectively. Finally, the association of sensitivities in compound-induced changes between phenotypic parameters and ion channel inhibitions measured using patch clamp recording was analyzed. Successful ranking of compound-induced sensitivity of cell lines was in lined with visual inspection of raw data.

The calcium-binding photoprotein clytin II: Expression of the preferred human codon-optimized clytin II gene in Chinese hamster ovary-K1 cells and its use in the G-protein-coupled receptor assays.

Clytin II (CLII) is a Ca2+-binding photoprotein and has been identified as an isotype of clytin I (CLI). CLII consists of apoCLII (an apoprotein) and 2-peroxide of coelenterazine (an adduct of molecular oxygen to coelenterazine), which is identical to the widely used Ca2+-binding photoprotein, aequorin (AQ). However, CLII triggered by Ca2+ exhibits a 4.5-fold higher maximum luminescence intensity (Imax) compared to both AQ and CLI, and it is approximately 5 times less sensitive to Ca2+ than AQ. To confirm the suitability of the preferred human codon-optimized CLII (pCLII) gene for cell-based G-protein-coupled receptor (GPCR) assays, a transformant stably expressing apoprotein of pCLII using the pCLII gene in the mitochondria of CHO-K1 cells was established and in situ regenerated pCLII in the cells were applied to the high-throughput screening system. An ATP-stimulated GPCR assay for endogenous P2Y purinergic receptors was confirmed using the established stable transformant.