Monitor Drug-Induced Calcium Flux in iPSC-Derived Cardiomyocytes

Fluo-4 meets the EVOS FL Auto 2 Imaging System and Celleste Image Analysis Software

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Pluripotent stem cells (PSCs) have the ability to differentiate into any one of the different cell types in the human body and are an important research tool for disciplines such as developmental biology, regenerative medicine, and oncology, to name a few [1,2]. The discovery that a terminally differentiated cell such as a dermal fibroblast can be reverted back to a stem cell–like state has opened up the possibility of growing patient-specific tissue and organs that originate from a patient’s own cells and therefore have less chance of rejection when transplanted [3,4]. Differentiation of induced pluripotent stem cells (iPSCs)—using specialized media with the appropriate growth and signaling factors—into complex cell culture models such as cardiomyocytes or dopaminergic neurons has opened up new avenues for basic research and translational medicine.

Role of calcium in cardiac action potentials

During an action potential, cardiomyocytes contract by membrane depolarization, which is caused by the rapid influx of calcium into the cell through voltage-gated calcium channels. Ion indicators that increase in fluorescence upon binding calcium, such as Invitrogen fluo-4 dye, are commonly used to measure cardiomyocyte action potentials and contraction rate. As calcium enters the cell, it binds to intracellular fluo-4, causing the fluorescence of the calcium indicator to increase. Likewise, the fluorescence of the indicator is reduced as intracellular calcium concentration decreases when calcium is pumped back out of the cell during the repolarization phase of the action potential [5-8].

Improper functioning of action potentials in cardiomyocytes can lead to a variety of human diseases such as hypertension. Drugs for treating high blood pressure, including the calcium channel blocker verapamil, act by helping to control cardiac action potentials. Furthermore, several compounds are produced by the body to increase the action potential rate in response to stimuli, such as the release of norepinephrine during the fight-or-flight response. Here we demonstrate a simple method for monitoring drug-induced changes in the rate of action potentials in human iPSC-derived cardiomyocytes.

Quantify calcium influx rate

To quantify the rate of calcium influx in iPSCs, we used the calcium indicator fluo-4 in a live-cell kinetic assay. Cell fluorescence was detected and analyzed on the Invitrogen EVOS FL Auto 2 Imaging System using Invitrogen Celleste Image Analysis Software. This live-cell imaging and analysis system enables quick and easy quantitative calcium imaging (Figure 1)—which is required when studying physiological responses to drugs in muscle cells, neurons, and other cell systems—without the need for specialized equipment or techniques.

The EVOS FL Auto 2 system was used to record videos of pulsing cardiomyocytes, allowing visualization of rapid calcium flux based on changes in the fluorescence intensity of the fluo-4 calcium indicator over time (Figure 1A). By simply selecting the “measure:intensity” tracking function after defining the region of interest in each video of contracting cardiomyocytes, Celleste software was used to generate a graph of the calcium flux and contraction rate based on fluorescence intensity changes observed (Figure 1B).

This function allowed quantitation of the baseline calcium flux and contraction rate in cardiomyocytes, along with changes in the pulse rate with different treatments. Application of 10 μM norepinephrine nearly doubled the rate of contraction compared with that seen in cardiomyocytes receiving no drug treatment (Figures 2A and 2B). Treatment with 50 nM verapamil reduced the rate of contraction to about half of the baseline cardiomyocyte contraction rate, whereas 1 μM verapamil prevented calcium flux, resulting in no contractions (Figures 2C and 2D).

Fluorescence imaging in real time

Drug-induced changes in cardiomyocyte calcium flux, measured with the fluorescent calcium indicator fluo-4, can easily be imaged and quantified using the EVOS FL Auto 2 Imaging System and Celleste Image Analysis Software. Real-time fluorescence videos of calcium flux in pulsing cardiomyocytes are recorded by selecting the “record video” option on the EVOS FL Auto 2 system, and then changes in fluorescence intensity are quantified by defining a region of interest and using the “measure:intensity” tracking function with the Celleste software. This simple quantitation system can be applied to cell physiology and drug discovery assays using other fluorescent indicators. EVOS microscopes incorporate high-resolution cameras, bright and digitally controlled LED light cubes, and interfaces structured for usability that allow the acquisition of stunning, publication-quality images and data.

References

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2. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. (1998) Science 282:1145–1147. 
3. Takahashi K, Yamanaka S (2006) Cell 126:663– 676. 
4. Menon T, Firth AL, Scripture-Adams DD et al. (2015) Cell Stem Cell 16:367–372. 
5. Guatimosim S, Guatimosim C, Song LS (2011) Methods Mol Biol 689:205–214. 
6. Itzhaki I, Rapoport S, Huber I et al. (2011) PLoS One 6:e18037. 
7. Eng G, Lee BW, Protas L et al. (2016) Nat Commun 7:10312. 
8. Ahola A, Pölönen RP, Aalto-Setälä K et al. (2018) Ann Biomed Eng 46:148–158.