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

AbstractIntroductionMulti-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.MethodsExtracellular 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.ResultsMany 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.DiscussionFP 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

Unravelling nicotinic receptor and ligand features underlying neonicotinoid knockdown actions on the malaria vector mosquito Anopheles gambiae

With the spread of resistance to long-established insecticides targeting Anopheles malaria vectors, understanding the actions of compounds newly identified for vector control is essential. With new commercial vector-control products containing neonicotinoids under development, we investigate the actions of 6 neonicotinoids (imidacloprid, thiacloprid, clothianidin, dinotefuran, nitenpyram and acetamiprid) on 13 Anopheles gambiae nicotinic acetylcholine receptor (nAChR) subtypes produced by expression of combinations of the Agα1, Agα2, Agα3, Agα8 and Agβ1 subunits in Xenopus laevis oocytes, the Drosophila melanogaster orthologues of which we have previously shown to be important in neonicotinoid actions. The presence of the Agα2 subunit reduces neonicotinoid affinity for the mosquito nAChRs, whereas the Agα3 subunit increases it. Crystal structures of the acetylcholine binding protein (AChBP), an established surrogate for the ligand-binding domain, with dinotefuran bound, shows a unique target site interaction through hydrogen bond formation and CH-N interaction at the tetrahydrofuran ring. This is of interest as dinotefuran is also under trial as the toxic element in baited traps. Multiple regression analyses show a correlation between the efficacy of neonicotinoids for the Agα1/Agα2/Agα8/Agβ1 nAChR, their hydrophobicity and their rate of knockdown of adult female An. gambiae, providing new insights into neonicotinoid features important for malaria vector control

Influence of Temperature on the Anti-allergic Activity of Fucoidan Extracted from Saccharina japonica

It has been ascertained in our laboratory that fucoidan, a polysaccharide contained in Saccharina japonica, shows anti-allergic activity through galectin 9 secretion in blood. A crude fucoidan fraction was chromatographically fractionated into three fractions using a Toyopearl-DEAE 650 M column in stepwise elusion with 0, 0.5 and 1.0 M NaCl in 0.05 M Tris-HCl buffer (pH 7.4). Each fraction was assessed using the passive cutaneous anaphylaxis (PCA) reaction. The non-absorbed fraction of the three fractions suppressed PCA, whereas the fractions eluted with 0.5 M and 1.0 M NaCl did not. Moreover, it was discovered that heat treatment of fucoidan at 50 °C for 10 min abolished its anti-allergic activity in the PCA reaction. Using DEAE chromatography, it was demonstrated that heat-treatment of the crude fucoidan fraction decreased the non-absorbed fraction, which possessed the anti-allergic activity, and increased the two fractions eluted with 0.5 M and 1.0 M NaCl. This clearly revealed the importance of temperature in maintaining the anti-allergic activity of fucoidan

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

<div><p>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.</p></div

Relationship between field potential duration (FPD)/corrected FPD (FPDc) and interspike interval (ISI) of 96 samples of hiPSC-CM.

<p>The data in sham treatment from 4 facilities were plotted (n = 96) in Fig 2a (FPD-ISI), Fig 2b (FPDcF-ISI) and Fig 2c (FPDcB-ISI), respectively. The solid and dashed lines indicate the linear regression line and 95% confidence bands, respectively. The equation, R² value, and root mean squared prediction error (RMSE) are shown in the figures.</p

Assay design and representative field potential (FP) waveforms before/after drug application.

<p>Schematic depicting the measurement schedule for the hiPSC-CM/MEA assay (Fig 1a). The upper panels show FP waveforms and parameters, and the lower panels indicate the arrhythmogenic waveform of EAD (left, dashed circle) and TA (right, dashed circle) (Fig 1b). Example FP waveforms after application of DMSO, E-4031, cisapride or chromanol 293B (Fig 1c).</p

Effects of <i>I</i>_Kr and <i>I</i>_Ks inhibitors on field potential duration (FPD)-interspike interval (ISI) plots.

<p>FPD-ISI plots are shown before and after compound application both in terms of absolute values (left) and % change (right), for DMSO (Fig 3a), E-4031 (Fig 3b), cisapride (Fig 3c) and chromanol 293B (Fig 3d). The solid lines and dashed lines indicate the linear regression lines and 95% confidence bands, respectively. Data for the middle concentrations are omitted for clarity. ‘Slope’ refers to the slope of the regression line.</p