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

Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

A single-molecule tracking technique coupled with mathematical modeling was developed for fully determining the dynamic monomer–dimer equilibrium of molecules in or on the plasma membrane, which will provide a framework for understanding signal transduction pathways initiated and regulated by dynamic dimers of membrane-localized receptors

Compensation, insurance, and management of injuries in investigator-initiated clinical trials in Japan

Introduction: The recent revision of the Declaration of Helsinki strengthened the ethical obligation to provide compensation for harm of research subjects. However, the actual status of providing compensation has rarely been disclosed, especially in the situation of investigator-initiated clinical trials (IITs).Objectives: To figure out the actual situation of compensation for injured subjects of IITs in Japan and to identify the necessary steps to fulfill the ethical obligation of compensation.Method: We conducted two types of surveys of compensation in IITs in Japan over the past five years. A: questionnaire survey delivered to 1,700 institutions on (1) number and paid amount of compensation cases; number and amount of contract fees paid to insurance companies; and (2) institutional management system of compensation cases. B: complementary survey to the item (1) of the survey A, at two institutions, on contract fees paid to and insurance paid from insurance companies.Result: In the survey A, we found only two cases of compensation provided, but could not find any serious cases claiming payment from insurance companies, although we could not exclude possibility of undisclosed cases. In the survey B, at the two institutions, a substantial amount of contract fees has been paid to insurance companies without claiming insurance payment; however, the total amount for each institution is less than that expected for one case of a serious injury. Also we found that there remain several points to improve the institutional management system of compensation in IITs in Japan.Conclusion: To provide appropriate compensation for injured subjects of IITs, we need to develop an institutional management system, along with a system to gather statistical information of compensation cases, to achieve proper resource allocation. This system should also provide education for investigators and support staff to manage injury cases

UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides

Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. in this study, to examine whether a nucleotide excision repair enzyme, Escherichia coli UvrABC, could suppress the mutations induced by oxidized deoxyribonucleotides in vivo, oxidized DNA precursors, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate, were introduced into uvrA, uvrB, and uvrC E. coli strains, and mutations in the chromosomal rpoB gene were analyzed. Unexpectedly, these oxidized DNA precursors induced mutations only slightly in the uvrA and uvrB strains. In contrast, effect of the uvrC-deficiency was not observed. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with H_{2}O_{2}, and the rpoB mutant frequencies were calculated. The frequency of the H_{2}O_{2}-induced mutations was increased in all of the strains tested; however, the increase was three- to four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Thus, UvrA and UvrB are involved in the enhancement, but not in the suppression, of the mutations induced by these oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage

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