March 15, 2012 (Vol. 32, No. 6)

Detection of Compound Effects on Beating Frequency Made Possible with New Instrument

A research team led by Bruno Becker, Ph.D., from AstraZeneca’s R&D bioscience department, CVGI, Molndal, Sweden, tested Roche’s xCELLigence Cardio Instrument (Figure 1) for label-free and noninvasive screening for cardiotoxic and arrhythmic effects. Their published study (Assay and Drug Development Technologies, Dec. 2011) tested whether impedance readings are a useful way to detect compound effects on the beating frequency of human cardiomyocytes derived from induced pluripotent stem cells (hiPS-CM) and mouse cardiomyocytes derived from embryonic stem cells (mESC-CM).

Pharmaceutical companies are continuously improving preclinical model systems to more precisely identify nontoxic therapeutic drug candidates. Despite this, attrition rates during the clinical phase of drug testing remain high. Drug safety and lack of drug efficacy are the two leading reasons.

Cardiotoxicity screening attempts to identify and characterize the significant side effects of new compounds on the heart. Many different model systems are used to assess the effect of compounds and predict their effect in humans. Cellular overexpression systems and complex in vivo models are widely used. Many of the cardiotoxicity screening systems are also used in the earlier stages of drug discovery to validate compounds and their targets.

Figure 1. The xCELLigence Cardio Instrument

More Reliable Screening

Stem cell-derived cardiomyocytes (SC-CMs) are a promising new tool for in vitro target identification, validation, and compound screening. Compounds that target ion channels and ion-channel regulators can also affect action potential characteristics, electrocardiogram parameters, and cardiac beating frequency.

Studying the beating frequency of spontaneously active SC-CMs can provide valuable information about how a new compound will act on cells in the intact heart. Recent findings show that using cardiomyocytes derived from embryonic stem cell (ESC) or induced pluripotent stem cells (iPS-SC) have great potential for cardiac safety screening. This approach is particularly attractive because techniques such as patch clamp and single-unit microelectrode recordings are technically challenging, invasive, and of low throughput.

Cardiac Safety Screening

The xCELLigence Cardio Instrument is an impedance-based cell monitoring system using high sampling rates of 12.9 milliseconds per data point to measure the contraction movements of cardiomyocytes plated in sensor wells.

After seeding, cardiomyocytes attach to interdigitated electrode surfaces at the bottom of each culture plate well (Figure 2). Cardiomyocyte contractions change cellular morphology and, possibly, cellular adhesion on the electrode surface, resulting in a measurable change in electrical impedance. Detecting this signal requires that the cells in the well are beating in synchrony. Individual signals that are not in phase with each other cancel each other out.

Dr. Becker and his colleagues showed that stem cell-derived cardiomyocytes form the functional syncytium of interconnected cardiac cells undergoing electrical signal transfer, producing synchronized measurable contractions.

Figure 2. Electrodes at the bottom of the E-Plate Cardio 96

While the current xCELLigence Cardio Instrument measures signals from one 96-well plate at a time, additional 96-plates were run in parallel to increase throughput (Figure 3). In the future, workflow automation could be used to perform the frequent media exchanges required to maintain high-level uniform rhythmic baseline beating patterns for the 14-day experiments. RTCA data sweeps were performed every 40 seconds. Recorded data was analyzed using the RTCA Cardio Software.

Commercially available hiPS-CM (iCells®) and mESC-CM (Cor.At®) purified by antibiotic resistance, were seeded into separate sensor plates at cell densities ranging from 5.6 k/well to 200 k/well (hiPS-CM), and 11 k/well to 400 k/well (mESC-CM), in wells coated with fibronectin, gelatin, or in uncoated wells.

Plating densities were based on total viable cell counts determined using the Roche Cedex Cell Analyzer. The average cell diameter for both cell types ranged from 9 to 17 μm. First regular beats appeared after four days for hiPS-CM. Consistent regular patterns were observed by 9–11 days. Therefore, the optimal time for testing compounds was about 12–14 days in culture for both cell types.

Figure 3. The E-Plate Cardio 96

Effect of Nine Reference Compounds

Autonomic Nervous System: Adrenergic stimulation using isoproterenol increased the beating rate between +64% to +85%. As expected, cholinergic stimulation using carbachol slowed beating between -18% and -11% at first and second addition (which differed in concentration, whereby the first addition was the lower and the second addition the higher concentration). Interestingly, mESC-CM reacted stronger to carbachol than hiPS-CM, with contractions blocked at first compound addition.

L-type calcium current: Using amlodipine to block the L-type calcium current stopped beating in human cells at second compound addition and in mouse cells already at first addition, suggesting concentrations were too high and immediately toxic.

T-type calcium current: Effect of blocking T-type calcium channels was tested using four different compounds. Mibefradil did not show any effect on the concentrations used in hiPS-CM, whereas contractions in mESC-CM were inhibited at the second addition. The higher sensitivity of mESC-CM toward T-type calcium channel blockers was confirmed using three additional compounds.

hERG current: This current plays an important role during repolarization of the action potential. hERG current block is a frequent side effect, which can prolong the QT interval resulting in lethal ventricular arrhythmia torsade de pointes. The E-4031 specific hERG channel blocker decreased beating rate and induced irregular beating at first compound addition.

Pacemaker current: This current plays a key role in the spontaneous beating of nodal cells. As expected, Zatebradine reduced beating rate in both CM models.

This research study showed that the xCELLigence Cardio Instrument has potential for 96-well throughput screening of compounds for effects on cardiac beating patterns.

Markus Schmitz, Ph.D. ([email protected]), is manager R&D, cellular analysis, at Roche Diagnostics. For life science research only. Not for use in diagnostic procedures. XCELLIGENCE is a trademark of Roche. E-PLATE is a registered trademark of ACEA Biosciences. Other brands or product names are trademarks of their respective holders.

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