To obtain additional information on possible toxicity of compounds that caused a decrease in the peak count, live cells were stained with the green-fluorescent viability dye calcein AM (Life Technologies) after compound treatment and beat measurement. Cells were imaged using the MiniMax Imaging Cytometer, an upgradable option for the SpectraMax i3 Platform. Several cell analysis methods are available in SoftMax Pro Software, including cell count, cell proliferation, and marker expression. Changes in cell viability due to compound toxicity were analyzed using the cell proliferation analysis protocol to measure the area covered by live, calcein AM-stained cells in each well.
Using the combined analysis of cardiomyocyte beating and viability imaging, compounds with differing effects on cells were identified. The average peak count for control wells was 12 peaks per 25 second read time, and wells in which peak count was significantly reduced could be easily distinguished by the software.
Some compounds were identified as generally cytotoxic, causing a significant reduction in peak count and a decrease in percent area covered by viable cells. Other compounds adversely affected cardiomyocyte beating without impacting cell viability, as evidenced by a low peak count and a high percentage of area covered by viable cells.
Figure 1 shows plate views of the data for both cardiac beating (top) and area covered by viable cells (bottom). Plate views offer an easy visual check for correlation between the effects of compounds on beating patterns and cell viability. In Figure 2, more detailed views of individual kinetic traces and representative images are shown for compounds with different effects on cardiomyocytes.
Several compounds that dramatically reduced the peak count were shown to have little or no effect on overall cell viability. For example, digoxin, a drug widely used in the treatment of arrhythmias, reduced peak count from the roughly 12 beats per 25-second reading down to one or two beats. However, cell covered area was 71% compared to 82% in DMSO-treated controls—not a significant reduction in cell viability. The dopamine inverse agonist haloperidol had a distinct effect on beating profile without impacting viability. Other compounds, like staurosporine, greatly reduced both cell viability and peak count (Figure 2).