Medicine
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In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes
Chapters
Summary June 22nd, 2020
This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.
Transcript
This technique enables clarification of the pathophysiology of cardiac diseases through investigation of the correlation between in vitro and in vivo parameters in animal models and humans. The main advantage of this technique is that it allows the study of deep myofilament function using very small biopsies or samples that were stored frozen. This technique allows assessing in vitro the impact of therapeutic interventions on myofilaments.
Before attempting an experiment, it is advisable to practice the cardiomyocyte extraction a few times to learn how to select, glue, and activate the cardiomyocytes with good size, striation, and shape. Before beginning the procedure, adjust the temperature of the testing apparatus in chamber to 15 degrees Celsius and turn on the force transducer and the motor. Thaw three to five micrograms of myocardial sample in a Petri dish containing 2.5 milliliters of relax ISO solution and use a scalpel to precisely cut the tissue into small pieces without causing unnecessary cell damage.
When all of the tissue has been cut, use a cut pipette tip to transfer the entire volume of solution and tissue fragments into a Potter-Elvehjem glass and use a grinder to mechanically disrupt the tissue at 30 to 40 rotations per minute. When a good cell suspension has been obtained, add 250 microliters of Triton to a 15 milliliter tube containing 2.25 milliliters of relax ISO and add the cell suspension to the resulting solution. Gently invert the tube three times to mix and let the tube sit at room temperature for one minute, followed by a four-minute incubation on ice.
At the end of the ice incubation, bring the final volume of the tube up to 15 milliliters with additional relax ISO and mix the tubes three times by inversion as demonstrated, then centrifuge the cells, then carefully remove almost all liquid except the last three milliliters of the supernatant. After the last wash out, remove the supernatant up to a volume of five to 10 milliliters of cell suspension. For skinned cardiomyocyte selection, add a drop of cell suspension onto a coverslip placed on top of a glass slide in an inverted microscope slide holder and use the 20X objective to select a single rod-shaped cardiomyocyte with a good striation pattern and size.
Use two hands to rotate the coverslip until the selected cardiomyocyte is positioned horizontally with its ends aligned with the needle of the force transducer and the motor and use a pipette tip to place a thin line of glue along the side of the coverslip. To glue the cell into place, immerse the needle tips of the force transducer and the motor into the glue line to create a glue halo around both tips and quickly move the needle tip of the force transducer down so that it glues to one edge of the cardiomyocyte. Repeat this procedure with the tip of the motor and the other extremity of the cell.
After five to eight minutes, move both micro manipulators simultaneously to lift the needles about 15 micrometers to avoid gluing the cell to the coverslip. To measure the active and passive forces and the calcium sensitivity, fill the first experimental well with 55 to 100 microliters of relaxing solution and fill the second experimental well with 55 to 100 microliters of activating solution. Using the camera software, set the region of interest to an area of the cardiomyocyte with a clear pattern of striation and set the sarcomere length to 2.2 micrometers.
Measure the distance between the two extremes of the cardiomyocyte and record the value as the myocyte length within the software. Next, move the needles slightly higher and gently move the microscope stage so that the cell moves from the coverslip to the well containing the relaxing solution on the back of the stage. Select the protocol that contains two cell shortening to occur when the cell is sequentially emerged in the calcium and relaxing solutions.
To elicit an isometric contraction, move the microscope stage so that the cardiomyocyte moves from the relaxing to the activating solution. Upon reaching the force plateau, begin recording the force data. After 10 seconds, switch the cell to the relaxing solution and record the data until the test stops.
To determine the calcium sensitivity, replace the activating solution with 55 to 100 microliters of each calcium solution and record the data as just demonstrated. At the end of the measurement, stretch the tips of the force transducer and motor to remove the needles from the cell and use a cotton swab soaked in acetone to carefully remove the glue halo from the needle tips. Although a certain degree of deterioration and force decrease is expected after prolonged experiments, the values of active tension within the functional permeabilized cardiomyocytes should be relatively stable.
Here, representative force traces of three out of eight force recordings needed to carry out a protocol of myofilament calcium sensitivity are shown. By transferring the cell to a well containing the activating solution, the cardiomyocytes starts to develop force until it reaches a plateau. After a quick slack test, the baseline values of zero force can be obtained.
The slope of the last part of this curve can be used to determine the value of the rate of force redevelopment, which is a measure of the apparent rate of crossbridge attachment and detachment. After transferring the cell back to a well containing the relaxing solution, the cell relaxes and its force drops. In addition to myofilament calcium sensitivity and the length-dependent activation measurements, the sarcomere length dependencies of passive tension and the sarcomere length dependencies of the calcium activated force per cross-sectional area and the calcium independent tension can be calculated.
Finally, it is important to note that the way that the cardiomyocytes are isolated significantly impacts the results. Using skinned cardiomyocytes to assess cardiac function in vitro is an important technique to clarify the changes occurring at the cell level as well as to study myofilament mechanisms in different physiological and pathological conditions. This method has the advantage of requiring a minimal amount of myocardial sample and allowing the use of cardiomyocytes from a wide range of species, cardiac locations, and pathologies.
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