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Calcium transient assay on the FLIPR Tetra HT Cellular Screening System

Ncardia's expertise in detecting potential cardiotoxic effects through Ca2+ transients at high-throughput level (up to 384-well) will support you in making decisions regarding the cardiac safety profile of your compounds at early stages of preclinical drug development. The FLIPR Tetra® High-Throughput Cellular Screening System (Molecular Devices) is capable of simultaneous fluid dispensing and signal detection and is therefore ideal for the fast detection of acute compound effects.

In an application note, we described the analysis of fluorescent dye-based calcium transients of Pluricyte Cardiomyocytes upon compound treatment using the Molecular Devices FLIPR system. This demonstrates how we can provide a highly relevant in vitro model to study the cardiac safety profile of your compounds at an early stage of drug development.

Read the full application note

Application

Cardiac safety assessment: study design

Case studies were performed to assess the effects of a set of known cardioactive compounds on Pluricyte Cardiomyocytes using the FLIPR Tetra High-Throughput Cellular Screening System. The system allows simultaneous dispensing of test compounds onto cells incubated with a fluorescent calcium dye and real-time measurement of changes in fluorescent calcium transients at high temporal resolution in 96-, 384- or 1536-well plate formats.

Signal peak parameters can be calculated based on a dynamic threshold and derivative analysis, and provide cardioactive compound response parameters, including peak frequency, peak amplitudes, and average peak width. This makes this assay extremely valuable for the high throughput evaluation of potential cardiotoxic effects of test compounds.

Results

hERG potassium channel blockers block the rapid component of the delayed rectifier outward potassium current (Ikr), thereby delaying the repolarization phase of the cardiomyocyte action potential. At higher concentrations, blocking of the hERG channel may lead to TdP-like arrhythmias. Figure 1 shows the effect of hERG channel blocker E4031 on the calcium transient fluorescent signal in cardiomyocytes.

L-type calcium channel agonists increase the calcium influx during the plateau phase (the phase between the depolarization and repolarization phase of a cardiomyocyte action potential). Moreover, as calcium is important for cardiomyocyte contraction, activation of the L-type calcium channel may lead to changes in cardiomyocyte contractility. Figure 1 shows how treatment of cardiomyocytes with the L-type calcium channel agonist Bay K8644 resulted in increased fluorescent calcium transient amplitude, increased peak width, and reduced peak frequency

L-type calcium channel antagonists inhibit the calcium influx into cardiomyocytes during the plateau phase (the phase between the depolarization and repolarization of a cardiomyocyte action potential). As calcium is important for cardiomyocyte contraction, inhibition of the L-type calcium channel may lead to decreased contractility. Figure 1 shows how treatment of cardiomyocytes with increasing doses of nifedipine results in gradually decreasing fluorescent calcium transient amplitudes.

Acute compound effects on the calcium transient fluorescent signals of Pluricyte Cardiomyocytes

Figure 1. Screenshot of acute compound effects on the calcium transient fluorescent signals of Pluricyte® Cardiomyocytes. The screenshot shows a 30 seconds time scale. The concentration of each compound increases from bottom to top and each concentration was analyzed in triplicate. DMSO and Pluricyte® Cardiomyocyte Medium (right columns) were used as negative controls. The measurement was performed directly after compound addition.

Conclusion

This application note shows how we can assess the effects of a set of cardioactive compounds on calcium transients using the FLIPR Tetra system. The high-throughput format of this platform makes this assay exceptionally well-suited for screening potential cardiotoxic effects of novel drug candidates at early stages of drug discovery.

Through our >10 years of expertise, our Calcium Transient assay services can support your Drug Discovery projects, providing you with detailed analyses to study the effects of your compounds. Results are highly relevant for studying cardiac safety or efficacy profile of compounds through several stages of your drug development projects.

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