This protocol is significant because it provides researchers with the skills to employ donor human hearts for preclinical physiological drug testing. This tissue utilizes both voltage and calcium dyes for the simultaneous characterization of cardiac action potentials and intracellular calcium transients in human tissue slices. To set up the optical mapping system, attach a tissue bath with a bottom polydimethylsiloxane gel layer to a perfusion system and circulate one liter of recovery solution at 37 degrees Celsius oxygenated with 100%oxygen through the perfusion system at a flow rate fast enough to maintain the temperature and to prevent the accumulation of bath perfusate.
Then use a target to adjust the focus and alignment of the two CMOS cameras and use a green LED light source with a 520 plus or minus five nanometer wavelength to excite the voltage sensitive and calcium indicator dyes simultaneously. Before obtaining tissue sections, mix frozen and liquid cardioplegia solution and place the heart tissue into the ice cold cardioplegia bath and identify the left ventricular free wall. Glue the pre-made agarose gel to the back of the metal tissue holders of the vibratome.
Cut one centimeter cubed blocks of tissue in cold cardioplegic solution and use topical skin adhesive to quickly mount the tissue blocks onto the metal tissue holders with the endocardial surfaces facing up. Transfer the metal holders into the vibratome bath of ice cold oxygenated slicing solution so that the tissues are completely submerged and move the blade to the front edge of the tissue. Turn on the vibratome to begin slicing with the preset parameters, discarding the first several slices until the blade reaches beyond the trabeculae into the smooth endocardial tissue.
When the experimental tissue has been reached, carefully transfer each slice into individual 100 micrometer nylon mesh cell strainers in a culture dish containing room temperature oxygenated Tyrode's recovery solution and cover the slices with mesh washers to keep the tissue slices from curling. When all of the slices have been collected, carefully transfer each slice into individual wells of a six-well plate containing three milliliters of PBS per well. Rinse the slice with gentle rocking and repeat this process three times with fresh sterile PBS before transferring the samples into individual wells of a six-well plate containing three milliliters of 37 degree Celsius culture medium per well.
Then place the plate onto a orbital shaker at 20 revolutions per minute in a humidified incubator at 37 degrees, 30%oxygen, and 5%carbon dioxide. After 20 minutes of incubation in recovery solution for freshly cut slices or immediately following culturing, carefully transfer the slice of interest to the perfusion system tissue bath and pin down the four corners to the gel layer while applying minimal stretch to the tissue. Allow the slices to rest in this bath with circulating recovery solution for approximately 10 minutes before adding 0.3 to 0.5 microliters of stock Blebbistatin to the reservoir of recovery solution.
While the slice is incubating in the Blebbistatin, reconstitute 30 microliters of the stock voltage sensitive dye in one milliliter of recovery solution at 37 degrees. After 10 minutes of Blebbistatin treatment, turn off the pumps and slowly load 0.2 to 0.3 milliliters of working dye solution onto the surface of the slice over a period of 30 seconds. After 90 seconds, turn on the pumps to wash out any excess dye.
After dying the slice with stock calcium indicator as just demonstrated, focus the cameras onto the slice and pace the slice at one hertz with a two millisecond pulse width duration at 1.5 times the amplitude of the predetermined pacing threshold. Place a coverslip over the tissue slice and illuminate the slice with the LED excitation light source. Then record the emitted voltage and calcium signals with the cameras at 1, 000 frames per second.
To condition the voltage and calcium optical mapping data, in the appropriate analysis software, click condition parameters and remove background. Then adjust the signal conditioning parameters to obtain optimal action potential and calcium transient traces. To calculate the conduction velocity, select activation map and enter a start and end time to encompass a single action potential in the trace.
Then click regional map and select the region of interest to display the activation map of the selected region. Next, select conduction velocity map and select the start and end times. Adjust the inter-pixel resolution values based on the setup as necessary.
Click generate vector map to select a region of interest and display the conduction velocity vectors within that region. The mean, median, standard deviation, and number of vectors included in the analysis as well as the average angle of propagation of the conduction velocity vectors in the selected region will be displayed. To calculate the action potential duration, select action potential duration, calcium transient duration map and select the start and end times to encompass one full action potential.
Then click regional action potential duration calculation to select a region of interest and to generate the action potential duration map. To determine the rise time, select rise time and select the start and end times to select the upstroke of one single action potential or calcium transient. Enter the start and end percentage values and click calculate to select the region of interest and to determine the mean, median, standard deviation, and number of pixels included in the analysis of rise time.
To determine the calcium decay, select calcium decay and enter the start and end times to encompass the entire decay portion of a single calcium transient signal. Then click calculate tau to select the region of interest and to determine the mean, median, standard deviation, and number of pixels included in the analysis of calcium decay time constant. In this representative experiment, the action potential and calcium transient traces were signal conditioned.
The activation times were determined for each pixel and isochronal maps of the activation times were plotted from the conditioned voltage and calcium traces. Conduction velocity vectors were calculated using the activation times and the known inter-pixel resolution. The calcium transient decay constant was measured by fitting a polynomial to the decaying portion of the calcium traces.
The action potential duration and calcium transient duration were measured as the time duration between the activation time and a specified percent of the re-polarization or calcium removal from the cytoplasm respectively. The rise times of the voltage and calcium traces were also measured and mapped. In this representative analysis, the effect of doxorubicin on cardiac conduction was tested resulting in a reduction of the transverse conduction velocity.
It is important to make sure that the slices are in complete cardioplegic arrest to minimize damage to the tissue during the slicing procedure and to obtain viable slices. At the end of the protocol, the slices can be frozen or fixed for molecular assessment. The mechanistic pathways involved in the electrophysiological phenomenon of interest can then be determined.
Development of this human cardiac slice model has allowed the study of the responses to treatments and disease in human heart tissue bridging the gap between animal and clinical studies. When working with human tissues while performing this technique, be sure to wear the appropriate PPE and to take any other necessary safety precautions.
