Cell culture conditions affect the ability of high content imaging assay to detect drug-induced changes in cellular parameters in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used for drug safety and efficacy testing with various techniques, including high content imaging (HCI). Upon drug treatment, a significant number of hiPSC-CMs grown in regular 96-well plates coated with fibronectin detached from the bottom of the plate, complicating data acquisition. Several cell culture configurations were tested to improve cell adherence, and the effects of these configurations on total cell number, separation of feature values between the negative (DMSO 0.

The anesthetized guinea pig: an effective early cardiovascular derisking and lead optimization model.

Introduction: In recent years, the anesthetized guinea pig has been used increasingly to evaluate the cardiovascular effects of drug-candidate molecules during lead optimization prior to conducting longer, more resource intensive safety pharmacology and toxicology studies. The aim of these studies was to evaluate the correlations between pharmacologically-induced ECG changes in the anesthetized cardiovascular guinea pig (CVGP) with ECG changes in conscious non-rodent telemetry models, human clinical studies and effects on key cardiac ion channels.

Methods: We compared the effects of 38 agents on ion channel inhibition to their ECG effects in the CVGP.

A HESI consortium approach to assess the human predictive value of non-clinical repolarization assays.

Drug-induced ventricular arrhythmia and Torsades de Pointes remain a serious public health issues in bringing safe new pharmaceuticals to the market place. Under the auspices of the International Life Science Institute (ILSI)-Health and Environmental Sciences Institute (HESI), a consortium involving representatives from pharmaceutical companies, regulatory agencies and opinion leaders from the scientific and medical research communities has been initiated. The objectives are (1) to assess the concordance between signals in non-clinical repolarization assays and clinical QT interval prolongation; (2) to investigate the mechanisms for any discrepancy identified between non-clinical and clinical results and to determine viable and successful alternative approaches to identify these compounds; and (3) to assess the proarrhythmic potential of such compounds.

The hERG channel and risk of drug-acquired cardiac arrhythmia: an overview.

This review summarizes current knowledge of the cardiac rapidly activating delayed rectifier potassium current (I(Kr)), and its connection to drug-acquired QT prolongation and the associated risk of ventricular arrhythmia and fibrillation. The molecular characterization of hERG as the structural correlate of I(Kr) and the link between inherited long QT and the KCNH2 gene (hERG), have facilitated mechanistic studies of drug-acquired QT prolongation. The development of high throughput assays to evaluate drug effects on hERG has provided an avenue to determine structure activity relations (SAR) within chemical series.

Improved throughput of PatchXpress hERG assay using intracellular potassium fluoride.

Blockade of the human ether-a-go-go-related gene (hERG) potassium channel, with a consequent possibility of QT prolongation and increased susceptibility to a characteristic polymorphic ventricular arrhythmia, torsade de pointes, is an important cause of withdrawal of drugs from the market. In the aftermath of recent drug withdrawals, regulatory agencies now require in vitro hERG screening of all pharmaceutical compounds that are targeted for human use. To minimize the potential for failure in later-stage drug development, many pharmaceutical and biotechnology companies have begun to use automated patch clamp systems with higher throughput than conventional manual patch-clamp techniques to conduct routine functional hERG screening during drug discovery and early development.

Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I Ks) activity.

A cardiac safety concern for QT prolongation and potential for pro-arrhythmia exists due to inhibition of the cardiac slowly activating delayed rectifier potassium current, I(Ks). Selective inhibitors of I Ks have been shown to prolong the QT interval in animal models. On the other hand, I Ks has been considered as a target for anti-arrhythmic therapy due to certain biophysical and pharmacological properties and its expression pattern in the heart.

Flunarizine is a highly potent inhibitor of cardiac hERG potassium current.

Flunarizine has been widely used for the management of a variety of disorders such as peripheral vascular diseases, migraine, and epilepsy. The majority of its beneficial effects have been attributed to its ability to inhibit voltage-gated Ca2+ channels in the low micromolar range, albeit non-selectively, as flunarizine has been shown to inhibit a variety of ion channels. We examined the effects of flunarizine on potassium currents through cardiac channels encoded by the human ether-a-go-go related gene (hERG) stably expressed in CHO cells.

Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern.

Introduction: In vitro evaluation of drug effects on hERG K(+) channels is a valuable tool for identifying potential proarrhythmic side effects in drug safety testing. Patch-clamp recording of hERG K(+) current in mammalian cells can accurately evaluate drug effects, but the methodology has not been standardized, and results vary widely. Our objective was to evaluate two potential sources of variability: the temperature at which recordings are performed and the voltage pulse protocol used to activate hERG K(+) channels expressed in HEK293 cells.

A novel mechanism for the store-operated calcium influx pathway.

Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by depletion of intracellular calcium stores. However, the exact molecular mechanism of such communication remains unclear. Recently, we demonstrated that native SOC channels can be activated by calcium influx factor (CIF) that is produced upon depletion of calcium stores, and showed that Ca(2+)-independent phospholipase A(2) (iPLA(2)) has an important role in the store-operated calcium influx pathway.

Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry.

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels.