Our protocol describes a reliable system for measuring contractility in adult human primary cardiomyocytes. Our system is a medium throughput, non-invasive optical recording method that continuously measures contractility from multiple cells in parallel, and enables real-time tracking of drug effects. Our methods support the discovery of novel molecules with the most desired profile for the correction of heart failure.
It also provide a preclinical approach for predicting drug-induced contractility risks. To begin, place a single glass cover slip in each well of an eight-well culture plate. Prepare five micrograms per milliliter of laminin by adding 800 microliters of the human recombinant laminin 521 stock to 7.2 milliliters of solution B and mix well.
Add 200 microliters of the diluted laminin solution to the center of the cover slip. Cover the plate and stack it in a four degrees Celsius refrigerator. Dilute dimethyl sulfoxide or DMSO 1000-fold in solution C to make a 0.1%DMSO vehicle solution.
To test the compound at one, 10, 100 and 1000-fold of its therapeutic exposure of 0.001 micromolar, dissolve the compound in DMSO at a one millimolar concentration. Dilute the DMSO test compound solution serially with DMSO to produce three further stocks. Finally, dilute each test compound stock 1000 old in 50 milliliters of solution C to obtain final test concentrations.
Add the vehicle solution and solutions of final micromolar concentrations of the compound to 50-milliliter syringes. Connect the syringes to the gravity flow system, then prime the gravity flow system. Remove an eight-well plate containing a laminate-coated cover slip out of the refrigerator and place one cover slip in a clean recording chamber.
Return the plate to the refrigerator until the next plating. Aspirate solution B from the escalated vial to reach the smallest volume without losing cells. Then dispense 200 microliters of the cell solution into the recording microscope chamber mounted on the stage of an inverted microscope and allow the cells to settle on the cover slip for five minutes.
Next, open the field of view on the microscope and determine if the cell density is adequate for the experimental run to begin. Once the plating is done, equilibrate the cells for five minutes by continuously perfusing them with solution C using the gravity flow perfusion system. Adjust the suction correctly, turn on the temperature control box and a heating plate and set them to deliver 35 degrees Celsius.
Then on a field stimulator with a pair of platinum wires placed on opposite sides of the chamber, stimulate the cells with suprathreshold voltage at a one hertz pacing frequency. Set the amplitude of the stimulating pulse at one volt and increase it until the cardiomyocytes start generating contraction-relaxation cycles. Select healthy cells with rod-shaped morphology and clear striations, then adjust the field of view and focus on bringing as many contracting cells as possible into view.
Next, displayed the digitized images of the cells within the acquisition software of the optical contractility recording system. While selecting the region of interest or ROIs, avoid out-of-focus areas and areas close to the edge of the cells. Initiate the experiment to evaluate the compound's effects.
The acquisition software will manage data acquisition. Display, and labeling of test concentrations and treatment time automatically. Apply test concentrations if the contraction remains at a stable amplitude throughout the entirety of the baseline vehicle period.
Disqualify cells displaying either a runup or rundown. Perform offline analysis using the analysis software and a custom-made macro to average the data. The software calculates and reports various metrics from the sarcomere dynamics data produced by the acquisition software.
Quantify the test compound effects on the average contractility amplitude relative to each cardiomyocyte's specific baseline vehicle control condition. Express the average results as mean plus/minus SEM and produce a graph plotting the concentration effect of the test compound on the contractility amplitude. Then fit the concentration response curve to the hill equation to derive IC50 and EC50 values.
Next, identify the aftercontraction as a spontaneous secondary contraction transient of the cardiomyocyte that occurs before the next regular contraction and produces an abnormal and unsynchronized contraction. Identify contraction failure as the inability of the electrical stimulus to induce a contraction. Visualize the short-term variability, or STV, and alternans in Poincare plots of contraction amplitude variability.
Calculate the STV with the last 20 transients of each control and test article concentration period. Then identify alternans as repetitive, alternating short and long contractility amplitude transients. To calculate the incidences of pro-arrhythmia, normalize the STV values to the vehicle control value of each cell, plot aftercontraction, contraction failure, STV and alternans and express them as a percent of the incidence of cells exhibiting each of the signals.
Complete the multi-parametric mechanistic profiling with the calculation of time to peak, decay to 30%then 90%relaxation, time to 90%relaxation, baseline sarcomere length, time to 50%peak, peak height, sarcomere length at peak contractility, maximum contraction velocity, and maximum relaxation velocity. Then express these parameters relative to each cardiomyocyte's specific baseline control condition and graph them in concentration response plots. This study shows human cardiomyocyte contractility measurement validation, and the effect of beta-adrenergic stimulation.
There were no spontaneous contractility transients in human cardiomyocytes, and the cardiomyocytes respond to external electrical stimulation with contractility-relaxation cycles, as well as to isoproterenol, a beta-adrenergic agonist. The isoproterenol produces a concentration-dependent increase in contractility, and its effects on the kinetics of contractility transient were also characterized. Following measurement of contractility, we can perform measurement of calcium transient with a fluorescent indicator.
Such data helps to determine if change in contractility require change In calcium dynamic. Our method paves the way for cardiac researchers to develop a better understanding of the physiology and pharmacology of human cardiomyocytes, establish structure, activity, and relationships of novel drugs, and explore their mechanism of action.
