Cardiomyocytes generated from Induced Pluripotent Stem or IPS cells are increasingly used to answer a variety of biological questions in the field of cardiac electrophysiology. The main advantages of this technology are that it allows a dramatic increase in throughput as well as subtype specific action potential recordings in ventricular like myocytes, using a ventricular specific promoter element. The introduction of genetically encoded voltage sensors through lentiviral transduction allows the optical imaging of human-induced pluripotent stem cells-derived cardiomyocytes action potentials.
Begin by mixing lentivirus-containing cell culture supernatant with cardiomyocyte maintenance medium, at a one to one ratio. Add hexadimetherine bromide at a final concentration of 8 micrograms per milliliter, to enhance the infection efficiency. And replace the cardiomyocyte maintenance medium in the IPS cell-derived cardiomyocytes culture dish, with the infection medium.
Culture the infected cardiomyocytes for 12 hours at 37 degrees Celsius, and replace the infection medium with fresh cardiomyocyte maintenance medium. Before imaging, exchange the cell culture medium with Tyrode's solution, supplemented with calcium. Then place the cardiomyocytes in an imaging chamber on the stage of an inverted epifluorescence microscope equipped with an image splitter.
If electrical pacing is required, install pacing electrodes in the imaging chamber and connect the electrodes to a stimulus generator. Bring the cells into focus, and locate a cell expressing the genetically encoded voltage indicator in the center of the viewing field. Set the optical path so that the emitted light is routed to the image splitter and switch the filter cube in the microscope to the one to be combined with the filters in the image splitter.
In the software controlling the camera, set the acquisition setting so that high-speed imaging is possible, and set up the image splitter according to the manufacturer's instructions. The two images representing the two different emission wavelength bands should be adjacent to each other, each occupying one half of the image. Check and adjust the focus of the image produced by the camera as necessary.
And adjust the brightness of the illumination light so that pixel saturation is avoided. Set the acquisition of the time series, and dim the light in the room to avoid stray light influencing the measurement. Then open the excitation light shutter and begin the acquisition of the image series, starting the electrical pacing at the appropriate time point, as experimentally appropriate.
When the acquisition is finished, close the excitation light shutter as necessary and save the recording to the hard drive. Then, leaving the recording setting unchanged, record the background over a region of the coverslip without cells. For analysis of the optical membrane potential recordings, open the tiff stack containing the cells in ImageJ, and use the free-hand selection tool to draw a region of interest over a fluorescent cardiomyocyte, in the red channel, taking care that the area is large enough that the cell will not move out of the region while contracting.
Next, select Analyze, Tools, Region Of Interest Manager and select Add T, to add the region as Region Of Interest 1. Drag the region of interest over the same cell in the green channel, and add this region as Region Of Interest 2. Under the Analyze and Set Measurements menus, unselect all of the options except for the Mean gray value.
In the Region Of Interest Manager, click More and Multi Measure to select the Measure all slices and One row per slice options and click OK.A window will open containing a spreadsheet with the three rows. Use File and Save As to save the spreadsheet into a comma separated value file. Close the window with the image stack and the spreadsheet window without closing the Region of Interest Manager window, and open the tiff stack containing the background measurement.
In the Region Of Interest Manager, click the More and Multi Measure to select the Measure all slices and One row per slice options and click OK.Then save the background data in a separate csv file. Here, a representative single IPS cell-derived cardiomyocyte is depicted with the white dotted lines marking the regions of interest, drawn during the imaging analysis in the RFP and GFP channels is shown. The signal from the RFP channel demonstrates a periodic increase in fluorescent intensity during each action potential due to an increasing forced or resonance energy transfer caused by changes in the membrane potential.
Concordantly, the GFP signal exhibits a periodic decrease in fluorescent intensity. The RFP/GFP ratio is the biological signal used in downstream analyses, such as action potential duration 50 and 90 measurements. For example, using this method, 30-day old ventricular like IPS cell-derived cardiomyocytes placed at 0.5 Hertz, showed a mean action potential duration 50 of 439 milliseconds, and a mean action potential duration 90 of 520 milliseconds.
Electrical stimulation of the IPS cell-derived cardiomyocytes at increasing stimulation rates every five beats, demonstrated a progressive shortening of the action potentials as the beating rate increased. Moreover, treatment of the spontaneously beating IPS cell-derived cardiomyocytes with isoproternol, a nonselective beta adreno receptor agonist, led to a prompt increase in the beating rate, that could be further increased with increasing doses of the agonist. While this method provides superior throughput compared to classical electrophysiological measurements, one must be aware of its limitations, including a temporal resolution that is limited by the intrinsic kinetic properties of the fluorescent voltage sensor.
