Cardiovascular (CV) toxicity is a leading contributor to drug withdrawal and late-stage attrition in the drug discovery process. There is an urgent need for novel in vitro assays that enable earlier and broader testing for CV activity.
The most relevant cells for pre-clinical testing are human cells. Use of these cells has the potential to add significant efficiencies, reduce animal use and decrease the number of dangerous drugs that reach the market.
Stem cell-derived human cardiomyocyte (SC-hCM) electrophysiology is a valuable tool to assess cardiac risk associated with drugs. Several studies have focused on the response of SC-hCMs to diverse types of drugs, using conventional electrophysiological approaches or impedance-based analysis of changes in the rhythmic beat patterns as end points. However, little attention has been put into the contraction itself, and the extent to which SC-hCM contraction is a surrogate for a “normal” contraction.
SC-hCMs show spontaneous contractile activity, resulting in transient changes in impedance (impedance twitches). Impedance-based measurement of SC-hCM contractile activity using the ACEA xCELLigence RTCA Cardio system adds a new dimension to cardiac risk assessment. The xCELLigence Cardio system measures the activity of SC-hCMs with time resolution sufficient to measure single contractions (sampling frequency = 77.5 Hz).
New Science being Pursued as a Marker for Safety
ChanTest has developed several algorithms for twitch detection, and kinetic analysis, and has concluded an investigation that suggests twitch impedance is a sensitive tool to assess contraction in a relatively high-throughput assay.
In the first pharmacologic study1, ChanTest investigated the mechanisms underlying impedance changes in iCELL® cardiomyocytes (provided by Cellular Dynamics International). These results suggest that several aspects of the excitation-contraction coupling mechanism, as observed in adult cardiomyocytes, are intact in SC-hCMs, including the dependence of the twitch contraction on Ca2+ entry (Figure 1) and the regulation of contraction by frequency and β-adrenergic stimulation.
Figure 1: Twitch Impedance is regulated by calcium entry. Transient impedance twitches from 3 wells (60 s sweeps/well) were overlayed and averaged (Mean ± SEM). The L-type calcium channel agonist FPL64176 (1 µM) increased twitch duration and amplitude, altered kinetics and slowed beat rate. Opposite effects were observed with the L-type calcium channel blocker verapamil (100 nM). The bottom right figure shows the concentration-dependence of the verapamil effect (delta Rel80 = change in duration at the 80% relaxation (decay) timepoint).
The study concluded that the impedance twitch recordings accurately reflect key elements of contraction, thus allowing the study of drugs that affect excitation-contraction coupling and the contractile apparatus. Importantly, the assay is conducted in a 96-well plate, thereby offering a significant throughput advantage over current animal-based methods for assessing contractility.
Finding Drug Effects that Are Not Detected using Conventional Electrophysiological Techniques
In a similar study2, ChanTest tested SC-hCMs for sensitivity to cardiotoxicants with longer-term (hours to days) mechanisms of action, effects that would not be detected using traditional electrophysiological methods. Cardiotoxic drugs, such the anthracycline cancer therapeutic doxorubicin, showed a clear decrease in impedance over time, consistent with cell death (Figure 2). In contrast, pentamidine, a drug that is known to induce cardiac arrhythmia by inhibiting hERG ion channel trafficking, did not reduce the overall impedance, but did induce expected changes in the twitch impedance (contraction; data not shown). Therefore, ChanTest's unique analysis can allow for sensitive detection of drug effects that may be missed by focusing on traditional endpoints alone.
Since impedance signals can be recorded over long periods of time, deleterious long-term drug effects, such as those commonly observed for oncology drugs, can be easily evaluated with this label-free procedure. The xCELLigence platform, applied to SC-hCMs, offers an effective non-invasive screen for the cardiac activity of drugs.
Figure 2: Long-term stability of the impedance signal (CI: Cell Index) allows detection of cardiotoxicity over days of exposure. The overall CI was normalized to zero, averaged (Mean ± SEM; n = 6 per condition) and plotted over 54 hours. The reduction in Normalized CI in the presence of doxorubicin is consistent with cell death. Vehicle control (0.1% DMSO) and pentamidine, which has long-term effects on the electrical properties of cardiomyocytes but is not cytotoxic, did not change the overall impedance.
If your goal is to more thoroughly evaluate the range of potential cardiotoxic effects of your compounds, contact us at ChanTest, so we can discuss these options for your drug discovery program.
1Carlos Obejero-Paz, Carlos, Andrew Bruening-Wright, Marina Kojukhova, and Arthur M. Brown. "EVALUATION OF CONTRACTILE ACTIVITY OF STEM CELL-DERIVED HUMAN CARDIOMYOCYTES USING IMPEDANCE ANALYSIS." ChanTest Corporation, Cleveland, OH, USA
2Bruening-Wright, Andrew, Carlos Obejero-Paz, Marina Kojukhova, and Arthur M. Brown. "Detection of Cardioactive Drug Effects Using a Plate-based Impedance Reader." ChanTest Corporation, Cleveland, OH, USA