The use of ratiometric fluorescence measurements of the voltage sensitive dye Di-4-ANEPPS to examine action potential characteristics and drug effects on human induced pluripotent stem cell-derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from 2 commercial sources (Cor.4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneously monitors 2 wavebands of emitted fluorescence, allowing ratiometric measurement of membrane voltage. Signals were acquired at 10 kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though the mean and variance differed significantly (APD90: 229 ± 15 ms vs 427 ± 49 ms [mean ± SD, P < 0.01]; average spontaneous cycle length: 0.99 ± 0.02 s vs 1.47 ± 0.35 s [mean ± SD, P < 0.01], Cor.4U vs iCell CMC, respectively). The 10–90% rise time of the AP (Trise) was ∼6 ms and was normally distributed when expressed as 1/T2riseTrise2 in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide a minimally invasive, quantitative, and accurate method to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations

Uncertainty quantification reveals the importance of data variability and experimental design considerations for in silico proarrhythmia risk assessment

The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a global initiative intended to improve drug proarrhythmia risk assessment using a new paradigm of mechanistic assays. Under the CiPA paradigm, the relative risk of drug-induced Torsade de Pointes (TdP) is assessed using an in silico model of the human ventricular action potential (AP) that integrates in vitro pharmacology data from multiple ion channels. Thus, modeling predictions of cardiac risk liability will depend critically on the variability in pharmacology data, and uncertainty quantification (UQ) must comprise an essential component of the in silico assay. This study explores UQ methods that may be incorporated into the CiPA framework. Recently, we proposed a promising in silico TdP risk metric (qNet), which is derived from AP simulations and allows separation of a set of CiPA training compounds into Low, Intermediate, and High TdP risk categories. The purpose of this study was to use UQ to evaluate the robustness of TdP risk separation by qNet. Uncertainty in the model parameters used to describe drug binding and ionic current block was estimated using the non-parametric bootstrap method and a Bayesian inference approach. Uncertainty was then propagated through AP simulations to quantify uncertainty in qNet for each drug. UQ revealed lower uncertainty and more accurate TdP risk stratification by qNet when simulations were run at concentrations below 5× the maximum therapeutic exposure (Cmax). However, when drug effects were extrapolated above 10× Cmax, UQ showed that qNet could no longer clearly separate drugs by TdP risk. This was because for most of the pharmacology data, the amount of current block measured was <60%, preventing reliable estimation of IC50-values. The results of this study demonstrate that the accuracy of TdP risk prediction depends both on the intrinsic variability in ion channel pharmacology data as well as on experimental design considerations that preclude an accurate determination of drug IC50-values in vitro. Thus, we demonstrate that UQ provides valuable information about in silico modeling predictions that can inform future proarrhythmic risk evaluation of drugs under the CiPA paradigm

Nonclinical cardiovascular safety of pitolisant: comparing International Conference on Harmonization S7B and Comprehensive in vitro Pro-arrhythmia Assay initiative studies

Background and purpose: We evaluated the concordance of results from two sets of nonclinical cardiovascular safety studies on pitolisant. Experimental approach: Nonclinical studies envisaged both in the ICH S7B guideline and Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were undertaken. CiPA-initiative studies included in vitro ion channels and stem cell-derived human ventricular myocyte studies as well as in silico modelling of results to simulate human ventricular electrophysiology. ICH S7B-recommended studies included in vitro hERG studies, in vivo dog study with follow-up investigations in rabbit Purkinje fibres and in vivo studies in the Carlsson rabbit proarrhythmia model. Key results: Both sets of nonclinical studies consistently excluded pitolisant from having clinically relevant QT-liability or proarrhythmic potential. CiPA studies revealed pitolisant to have modest calcium channel blocking and late I Na reducing activities at high concentrations, which resulted in reduction of dofetilide-induced early after-depolarisations (EADs) by pitolisantin ICH S7B studies. Studies in stem cell-derived human cardiomyocytes with dofetilide or E-4031 given alone and in combination with pitolisant confirmed these properties. In silico modelling confirmed that the measured ion channel effects are consistent with results from both the stem cell-derived cardiomyocyte and rabbit Purkinje fibre studies and categorised pitolisant as a drug with low torsadogenic potential. The results from the two sets of nonclinical studies correlated well with two clinical QT studies. Conclusions and implications: Our experience supports the CiPA initiative but suggests that sponsors should consider investigating drug effects on EADs and the use of proarrhythmia models when the results from CiPA studies are ambiguous

Electrophysiological characterization of drug response in hSC-derived cardiomyocytes using voltage-sensitive optical platforms

Introduction: Voltage-sensitive optical (VSO) sensors offer a minimally invasive method to study the time course of repolarization of the cardiac action potential (AP). This Comprehensive in vitro Proarrhythmia Assay (CiPA) cross-platform study investigates protocol design and measurement variability of VSO sensors for preclinical cardiac electrophysiology assays. Methods: Three commercial and one academic laboratory completed a limited study of the effects of 8 blinded compounds on the electrophysiology of 2 commercial lines of human induced pluripotent stem-cell derived cardiomyocytes (hSC-CMs). Acquisition technologies included CMOS camera and photometry; fluorescent voltage sensors included di-4-ANEPPS, FluoVolt and genetically encoded QuasAr2. The experimental protocol was standardized with respect to cell lines, plating and maintenance media, blinded compounds, and action potential parameters measured. Serum-free media was used to study the action of drugs, but the exact composition and the protocols for cell preparation and drug additions varied among sites. Results: Baseline AP waveforms differed across platforms and between cell types. Despite these differences, the relative responses to four selective ion channel blockers (E-4031, nifedipine, mexiletine, and JNJ 303 blocking IKr, ICaL, INa, and IKs, respectively) were similar across all platforms and cell lines although the absolute changes differed. Similarly, four mixed ion channel blockers (flecainide, moxifloxacin, quinidine, and ranolazine) had comparable effects in all platforms. Differences in repolarisation time course and response to drugs could be attributed to cell type and experimental method differences such as composition of the assay media, stimulated versus spontaneous activity, and single versus cumulative compound addition. Discussion: In conclusion, VSOs represent a powerful and appropriate method to assess the electrophysiological effects of drugs on iPSC-CMs for the evaluation of proarrhythmic risk. Protocol considerations and recommendations are provided toward standardizing conditions to reduce variability of baseline AP waveform characteristics and drug responses

Separation of early afterdepolarizations from arrhythmogenic substrate in the isolated perfused hypokalaemic murine heart through modifiers of calcium homeostasis

In human type 1 diabetes (T1D) and in its murine model, the major histocompatibility complex (MHC) class II molecules, human leukocyte antigens (HLA)-DQ and -DR and their murine orthologues, IA and IE, are the major genetic determinants. In this report, we have ranked HLA class II molecule-associated T1D risk in a two-sided gradient from very high to very low. Very low risk corresponded to dominant protection from T1D. We predicted the protein structure of DQ by using the published crystal structures of different allotypes of the murine orthologue of DQ, IA. We discovered marked similarities both within, and cross species between T1D protective class II molecules. Likewise, the T1D predisposing molecules showed conserved similarities that contrasted with the shared patterns observed between the protective molecules. We also found striking inter-isotypic conservation between protective DQ, IA allotypes and protective DR4 subtypes. The data provide evidence for a joint action of the class II peptide-binding pockets P1, P4 and P9 in disease susceptibility and resistance with a main role for P9 in DQ/IA and for P1 and P4 in DR/IE. Overall, these results suggest shared epitope(s) in the target autoantigen(s), and common pathways in human and murine T1D

Connect and Conquer: Collectivized Behavior of Mitochondria and Bacteria

The connectedness of signaling components in network structures is a universal featureof biologic information processing. Such organization enables the transduction ofcomplex input stimuli into coherent outputs and is essential in modulating activities asdiverse as the cooperation of bacteria within populations and the dynamic organizationof mitochondria within cells. Here, we highlight some common principles that underpincollectivization in bacteria and mitochondrial populations and the advantages conferredby such behavior. We discuss the concept that bacteria and mitochondria act as signaltransducers of their localized metabolic environments to bring about energy-dependentclustering to modulate higher-order function across multiple scales

Utility of Zebrafish Models of Acquired and Inherited Long QT Syndrome

Long-QT Syndrome (LQTS) is a cardiac electrical disorder, distinguished by irregular heart rates and sudden death. Accounting for ∼40% of cases, LQTS Type 2 (LQTS2), is caused by defects in the Kv11.1 (hERG) potassium channel that is critical for cardiac repolarization. Drug block of hERG channels or dysfunctional channel variants can result in acquired or inherited LQTS2, respectively, which are typified by delayed repolarization and predisposition to lethal arrhythmia. As such, there is significant interest in clear identification of drugs and channel variants that produce clinically meaningful perturbation of hERG channel function. While toxicological screening of hERG channels, and phenotypic assessment of inherited channel variants in heterologous systems is now commonplace, affordable, efficient, and insightful whole organ models for acquired and inherited LQTS2 are lacking. Recent work has shown that zebrafish provide a viable in vivo or whole organ model of cardiac electrophysiology. Characterization of cardiac ion currents and toxicological screening work in intact embryos, as well as adult whole hearts, has demonstrated the utility of the zebrafish model to contribute to the development of therapeutics that lack hERG-blocking off-target effects. Moreover, forward and reverse genetic approaches show zebrafish as a tractable model in which LQTS2 can be studied. With the development of new tools and technologies, zebrafish lines carrying precise channel variants associated with LQTS2 have recently begun to be generated and explored. In this review, we discuss the present knowledge and questions raised related to the use of zebrafish as models of acquired and inherited LQTS2. We focus discussion, in particular, on developments in precise gene-editing approaches in zebrafish to create whole heart inherited LQTS2 models and evidence that zebrafish hearts can be used to study arrhythmogenicity and to identify potential anti-arrhythmic compounds