Comprehensive in vitro cardiac safety assessment using human stem cell technology: Overview of CSAHi HEART initiative.

Recent increasing evidence suggests that the currently-used platforms in vitro IKr and APD, and/or in vivo QT assays are not fully predictive for TdP, and do not address potential arrhythmia (VT and/or VF) induced by diverse mechanisms of action. In addition, other cardiac safety liabilities such as functional dysfunction of excitation-contraction coupling (contractility) and structural damage (morphological damage to cardiomyocytes) are also major causes of drug attrition, but current in vitro assays do not cover all these liabilities. We organized the Consortium for Safety Assessment using Human iPS cells (CSAHi; http://csahi.org/en/), based on the Japan Pharmaceutical Manufacturers Association (JPMA), to verify the application of human iPS/ES cell-derived cardiomyocytes in drug safety evaluation. The main goal of the CSAHi HEART team has been to propose comprehensive screening strategies to predict a diverse range of cardiotoxicities by using recently introduced platforms (multi-electrode array (MEA), patch clamp, cellular impedance, motion field imaging [MFI], and Ca transient systems) while identifying the strengths and weaknesses of each. Our study shows that hiPS-CMs used in these platforms have pharmacological responses more relevant to humans in comparison with existent hERG, APD or Langendorff (MAPD/contraction) assays, and not only MEA but also other methods such as impedance, MFI, and Ca transient systems would offer paradigm changes of platforms for predicting drug-induced QT risk and/or arrhythmia or contractile dysfunctions. Furthermore, we propose a potential multi-parametric platform in which field potential (MEA)-Ca transient-contraction (MFI) could be evaluated simultaneously as an ideal novel platform for predicting a diversity of cardiac toxicities, namely whole effects on the excitation-contraction cascade.

A new paradigm for drug-induced torsadogenic risk assessment using human iPS cell-derived cardiomyocytes.

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are anticipated to be a useful tool for conducting proarrhythmia risk assessments of drug candidates. However, a torsadogenic risk prediction paradigm using hiPSC-CMs has not yet been fully established. METHODS: Extracellular field potentials (FPs) were recorded from hiPSC-CMs using the multi-electrode array (MEA) system. The effects on FPs were evaluated with 60 drugs, including 57 with various clinical torsadogenic risks. Actual drug concentrations in medium were measured using the equilibrium dialysis method with a Rapid Equilibrium Dialysis device. Relative torsade de pointes (TdP) scores were determined for each drug according to the degree of FP duration prolongation and early afterdepolarization occurrence. The margins were calculated from the free concentration in medium and free effective therapeutic plasma concentration. Each drug's results were plotted on a two-dimensional map of relative TdP risk scores versus margins. RESULTS: Each drug was categorised as high, intermediate, or low risk based on its location within predefined areas of the two-dimensional map. We categorised 19 drugs as high risk; 18 as intermediate risk; and 17 as low risk. We examined the concordance between our categorisation of high and low risk drugs against the torsadogenic risk categorisation in CredibleMeds®. Our system demonstrated high concordance, as reflected in a sensitivity of 81%, specificity of 87%, and accuracy of 83%. DISCUSSION: These results indicate that our torsadogenic risk assessment is reliable and has a potential to replace the hERG assay for torsadogenic risk prediction, however, this system needs to be improved for the accurate of prediction of clinical TdP risk. Here, we propose a novel drug induced torsadogenic risk categorising system using hiPSC-CMs and the MEA system.

Electrophysiological Characteristics of Human iPSC-Derived Cardiomyocytes for the Assessment of Drug-Induced Proarrhythmic Potential.

The aims of this study were to (1) characterize basic electrophysiological elements of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that correspond to clinical properties such as QT-RR relationship, (2) determine the applicability of QT correction and analysis methods, and (3) determine if and how these in-vitro parameters could be used in risk assessment for adverse drug-induced effects such as Torsades de pointes (TdP). Field potential recordings were obtained from commercially available hiPSC-CMs using multi-electrode array (MEA) platform with and without ion channel antagonists in the recording solution. Under control conditions, MEA-measured interspike interval and field potential duration (FPD) ranged widely from 1049 to 1635 ms and from 334 to 527 ms, respectively and provided positive linear regression coefficients similar to native QT-RR plots obtained from human electrocardiogram (ECG) analyses in the ongoing cardiovascular-based Framingham Heart Study. Similar to minimizing the effect of heart rate on the QT interval, Fridericia's and Bazett's corrections reduced the influence of beat rate on hiPSC-CM FPD. In the presence of E-4031 and cisapride, inhibitors of the rapid delayed rectifier potassium current, hiPSC-CMs showed reverse use-dependent FPD prolongation. Categorical analysis, which is usually applied to clinical QT studies, was applicable to hiPSC-CMs for evaluating torsadogenic risks with FPD and/or corrected FPD. Together, this results of this study links hiPSC-CM electrophysiological endpoints to native ECG endpoints, demonstrates the appropriateness of clinical analytical practices as applied to hiPSC-CMs, and suggests that hiPSC-CMs are a reliable models for assessing the arrhythmogenic potential of drug candidates in human.

A human ventricular myocyte model with a refined representation of excitation-contraction coupling.

Cardiac Ca(2+)-induced Ca(2+) release (CICR) occurs by a regenerative activation of ryanodine receptors (RyRs) within each Ca(2+)-releasing unit, triggered by the activation of L-type Ca(2+) channels (LCCs). CICR is then terminated, most probably by depletion of Ca(2+) in the junctional sarcoplasmic reticulum (SR). Hinch et al. previously developed a tightly coupled LCC-RyR mathematical model, known as the Hinch model, that enables simulations to deal with a variety of functional states of whole-cell populations of a Ca(2+)-releasing unit using a personal computer. In this study, we developed a membrane excitation-contraction model of the human ventricular myocyte, which we call the human ventricular cell (HuVEC) model. This model is a hybrid of the most recent HuVEC models and the Hinch model. We modified the Hinch model to reproduce the regenerative activation and termination of CICR. In particular, we removed the inactivated RyR state and separated the single step of RyR activation by LCCs into triggering and regenerative steps. More importantly, we included the experimental measurement of a transient rise in Ca(2+) concentrations ([Ca(2+)], 10-15 µM) during CICR in the vicinity of Ca(2+)-releasing sites, and thereby calculated the effects of the local Ca(2+) gradient on CICR as well as membrane excitation. This HuVEC model successfully reconstructed both membrane excitation and key properties of CICR. The time course of CICR evoked by an action potential was accounted for by autonomous changes in an instantaneous equilibrium open probability of couplons. This autonomous time course was driven by a core feedback loop including the pivotal local [Ca(2+)], influenced by a time-dependent decay in the SR Ca(2+) content during CICR.

Improvement of acquisition and analysis methods in multi-electrode array experiments with iPS cell-derived cardiomyocytes.

INTRODUCTION: Multi-electrode array (MEA) systems and human induced pluripotent stem (iPS) cell-derived cardiomyocytes are frequently used to characterize the electrophysiological effects of drug candidates for the prediction of QT prolongation and proarrhythmic potential. However, the optimal experimental conditions for obtaining reliable experimental data, such as high-pass filter (HPF) frequency and cell plating density, remain to be determined. METHODS: Extracellular field potentials (FPs) were recorded from iPS cell-derived cardiomyocyte sheets by using the MED64 and MEA2100 multi-electrode array systems. Effects of HPF frequency (0.1 or 1Hz) on FP duration (FPD) were assessed in the presence and absence of moxifloxacin, terfenadine, and aspirin. The influence of cell density on FP characteristics recorded through a 0.1-Hz HPF was examined. The relationship between FP and action potential (AP) was elucidated by simultaneous recording of FP and AP using a membrane potential dye. RESULTS: Many of the FP waveforms recorded through a 1-Hz HPF were markedly deformed and appeared differentiated compared with those recorded through a 0.1-Hz HPF. The concentration-response curves for FPD in the presence of terfenadine reached a steady state at concentrations of 0.1 and 0.3µM when a 0.1-Hz HPF was used. In contrast, FPD decreased at a concentration of 0.3µM with a characteristic bell-shaped concentration-response curve when a 1-Hz HPF was used. The amplitude of the first and second peaks in the FP waveform increased with increasing cell plating density. The second peak of the FP waveform roughly coincided with AP signal at 50% repolarization, and the negative deflection at the second peak of the FP waveform in the presence of E-4031 corresponded to early afterdepolarization and triggered activity. DISCUSSION: FP can be used to assess the QT prolongation and proarrhythmic potential of drug candidates; however, experimental conditions such as HPF frequency are important for obtaining reliable data.

Role of Mg(2+) block of the inward rectifier K(+) current in cardiac repolarization reserve: A quantitative simulation.

Different K(+) currents serve as "repolarization reserve" or a redundant repolarizing mechanism that protects against excessive prolongation of the cardiac action potential and therefore arrhythmia. Impairment of the inward rectifier K(+) current (I(K1)) has been implicated in the pathogenesis of cardiac arrhythmias. The characteristics of I(K1) reflect the kinetics of channel block by intracellular cations, primarily spermine (a polyamine) and Mg(2+), whose cellular levels may vary under various pathological conditions. However, the relevance of endogenous I(K1) blockers to the repolarization reserve is still not fully understood in detail. Here we used a mathematical model of a cardiac ventricular myocyte which quantitatively reproduces the dynamics of I(K1) block to examine the effects of the intracellular spermine and Mg(2+) concentrations, through modifying I(K1), on the action potential repolarization. Our simulation indicated that an I(K1) transient caused by relief of Mg(2+) block flows during early phase 3. Increases in the intracellular spermine/Mg(2+) concentration, or decreases in the intracellular Mg(2+) concentration, to levels outside their normal ranges prolonged action potential duration by decreasing the I(K1) transient. Moreover, reducing both the rapidly activating delayed rectifier current (I(Kr)) and the I(K1) transient caused a marked retardation of repolarization and early afterdepolarization because they overlap in the voltage range at which they flow. Our results indicate that the I(K1) transient caused by relief of Mg(2+) block is an important repolarizing current, especially when I(Kr) is reduced, and that abnormal intracellular free spermine/Mg(2+) concentrations may be a missing risk factor for malignant arrhythmias in I(Kr)-related acquired (drug-induced) and congenital long QT syndromes.

Molecular and functional characterization of ERG, KCNQ, and KCNE subtypes in rat stomach smooth muscle.

Contribution of K(+) channels derived from the expression of ERG, KCNQ, and KCNE subtypes, which are responsible for rapidly and slowly activating delayed rectifier K(+) currents (I(Kr) and I(Ks), respectively) in cardiac myocytes, to membrane currents was examined in stomach circular smooth muscle cells (SMCs). The region-qualified multicell RT-PCR showed that ERG1/KCNE2 transcripts were expressed in rat stomach fundus and antrum SMCs and that KCNQ1/KCNE1 transcripts were expressed in antrum but not fundus. Western blotting and immunocytochemical analyses indicate that ERG1 proteins were substantially expressed in both regions, whereas KCNE1 proteins were faintly expressed in antrum and not in fundus. Both I(Kr)- and I(Ks)-like currents susceptible to E-4031 and indapamide, respectively, were identified in circular SMCs of antrum but only I(Kr)-like current was identified in fundus. It is strongly suggested that I(Kr)- and I(Ks)-like currents functionally identified in rat stomach SMCs are attributable to the expression of ERG1/KCNE2 and KCNQ1/KCNE1, respectively. The membrane depolarization by 1 microM E-4031 indicates the contribution of K(+) channels encoded by ERG1/KCNE2 to the resting membrane potential in stomach SMCs.