This protocol can provide high throughput measurements of cellular electrophysiology in a manner that befits the rapidly developing technology of induced pluripotent stem cell-derived cardiomyocytes. The main advantage of this technique is that it is less complex and less labor-intensive than the traditional patch clamp technique, yet it is able to produce equivalent results. So this protocol can be expanded for large-scale toxicity screening programs using human induced pluripotent stem cell-derived cardiomyocytes.
For example, the comprehensive in vitro proarrhythmia assay. To prepare induced pluripotent stem cell-derived cardiomyocyte cultures for the experiment, first coat individual 10 millimeter round glass number zero cover slips with 150 microliters of 1:60 basement membrane matrix within each well of a 24-well plate, and incubate the cover slips at four degrees Celsius for four hours. At the end of the incubation, remove the excess matrix from the cover slips and apply the appropriate volume of cells to each cover slip.
After one hour at 37 degrees Celsius, carefully fill each well with plating medium and return the plate to the incubator. To prepare for voltage sensitive dye imaging, equip an inverted epifluorescence microscope with a 40x magnification high numerical aperture lens, and couple a fast switching warm white LED to the transmitted illumination port. Insert a simple red 660 nanometer filter into the transmitted light path and mount a fast switching 470 nanometer LED head for photometry recording.
Insert a 470/40 excitation filter at the epifluorescence port to clean up the light generated by the LED, and insert a microscope cube containing a 495 nanometer long pass beam splitter in the mirror unit carousel within the microscope. Fit a detection arm containing an adjustable field diaphragm to the C mount port to allow region of interest selection, and couple a PMT detector and a USB camera to the microscope. Insert a filter cube containing a 565 nanometer long pass beam splitter and a 535/50 emission filter into the PMT port to split the emission light between the two detectors.
Couple the PMT to a power supply and a PMT amplifier, and connect the PMT amplifier output to an analog input pin of a data acquisition system. To fulfill Nyquist criteria and to prevent aliasing, filter the analog data from the PMT at one kilohertz or higher, and digitize the data at a frequency that is at least double that of the highest frequency present in the analog signal. To load the cells with voltage sensitive dye, mix five microliters of 1000x FluoVolt and 50 microliters of power load solution, and add three microliters of the resulting loading solution to three milliliters of warmed Tyrode solution in a petri dish.
Add a single induced pluripotent stem cell-derived cardiomyocyte coated cover slip to the dish and place the dish at 37 degrees Celsius for 20 minutes. Mount a heated live cell imaging chamber onto the microscope stage and fill the chamber with 500 microliters of fresh Tyrode solution. Then wash the cover slip with fresh Tyrode solution at 37 degrees Celsius and use fine point forceps to carefully transfer the cover slip to the pre-warmed bath chamber.
To standardize the cellular dynamics and experimental parameters, insert two platinum electrodes into the chamber, spaced five millimeters apart, and connect the electrodes to an external stimulator. Set the stimulator to five millisecond bipolar field pulses at 0.5 hertz, and increase the stimulus from one volt until the cells begin to contract. Then set the voltage to roughly 25%above this threshold.
For optical action potential acquisition, use the transmitted light path and the USB camera to visualize the myocytes under brightfield conditions. Select an isolated cell and use the field diaphragm to tightly crop its optical path, ensuring that only light from the cell of interest is monitored. Activate the PMT amplifier, and set the PMT supply to 750 volts.
Run the stimulation protocol along with the acquisition software while simultaneously activating the 470 nanometer excitation light. Record 10 sweeps, ensuring that stable action potentials are detected. After the last sweep, immediately move the microscope stage while still recording to briefly acquire background signal from a region devoid of cells and turn off the excitation light.
To analyze the action potential data, open a saved recording in the appropriate analysis software and average 10 sweeps containing stimulated action potentials from a single cell. Subtract the mean of the baseline signal representing fluorescence offset from the average trace, then use the formula to calculate the change in fluorescence using diastolic fluorescence as F0.Identify the diastolic and action potential areas of interest and measure the desired cardiac action potential parameters, including, but not limited to, the duration at 50%and 90%repolarization. Then export the data from this single cell to a spreadsheet program.
This method allows for rapid and precise quantification of repolarization mechanics, which can provide valuable insights into cellular ionic abnormalities. Pronounced phase one features can be observed in optical signals from murine cardiomyocytes, which are morphologically distinct from those of human induced pluripotent stem cell-derived cardiomyocytes. In addition, human induced pluripotent stem cell-derived cardiomyocytes are responsive to pharmacological manipulation with nifedipine, a known L-type calcium channel antagonist.
During continuous drug application, a 41.5%decrease in action potential duration at 90%repolarization was observed, suggesting the functionality of fluorescent voltage indicator based imaging as a platform for prospective high throughput cardiac drug screening studies. It is important to keep in mind that although the voltage sensitive dye used in this study is less cytotoxic than others, the excitation light should still be used sparingly to conserve cellular viability. The modulatory photometry equipment used in this protocol is incredibly versatile.
For example, it can be used for simultaneous measurements of other electrophysiological parameters, such as intracellular calcium.
