The overall goal of this procedure is to isolate viable cardiomyocytes from disease human ventricular specimens and characterize their functional alterations. This is accomplished by first rapidly collecting and mincing fresh ventricular samples in ice cold cardioplegic solution in order to avoid excessive tissue degradation and cell damage. The second step is to perform a stepwise digestion of the myocardial chunks using a custom made digestion device and enzymatic solutions.
In six cycles, the cell containing buffer is collected in a tube at each cycle and diluted with a cell preserving solution. The final step is to resuspend the cells contained in the six tubes in a lower volume of standard physiological solution with normal calcium concentrations in order to perform the functional characterization. Ultimately, patch clamp recording of action potentials and simultaneous assessment of intercellular calcium using fluorescent dyes is used to show the alterations of action, potential duration, and intracellular calcium cycling in cardiomyocytes from patients with hypertrophic cardiomyopathy.
The main advantage of DYS unique over a 16 method, like the standard chunk dejection method is that in a association with enzyme solution, we use a custom made digestion device, which allow a joint de association of variable cytes thanks to its silicon scrapes. This method can help answer key questions in the field of cardiology, such as how the well-known molecular and structural remodeling associated with cardiac diseases reflects into alteration of single cardiomyocytes that can be analyzed by our method and targeted by specific drugs. So the implication of this technique extend toward the therapy of genetic cardiac diseases such as hypertrophic cardiomyopathy.
In fact, this method can be used to test novel neuropathic approaches on ventricular cardiomyocytes from patients with hypertrophic or ischemic cardiac diseases who underwent heart surgery. We first had the idea for this method when comparing the analogy between human artery specimens and ventricular biopsies obtained during cardiac surgery. Begin this procedure by pouring 40 milliliters of Cardioplegic solution in a 50 milliliter tube and store it in ice for specimen transportation from the operative room to the cell isolation lab rapidly transfer the specimen to the lab area and start specimen processing within 10 minutes from specimen excision.
Next collective ventricular myocardial specimen from the operative room immediately after excision. Wash it with ice cold CP solution and store it in the tube while keeping the specimen in ice cold CP buffer. Carefully remove the endocardial fibrotic layer using fine scissors afterwards.
Cut the myocardial tissue into two to three millimeter long small pieces with the total amount of ventricular myocardium between 100 milligrams and one gram for each isolation. After the chunks are transferred into the scraping chamber of the digestion device, change the CP buffer in the chamber with cold calcium free dissociation buffer. Then place the digestion device in a thermostatic bath.
Set the bath the 37.5 degrees Celsius and turn it on. Next turn on the motor of the digestion device and set the rotation speed to one revolution per second. Perform three washing cycles with DB and change the solution in the chamber with 37 degrees Celsius oxygen saturated clean DB every eight minutes.
After that, prepare enzyme buffer one by adding 250 units per milliliter of collagenase type five and four units per milliliter protease. Type 24 to the DB solution. Then prepare enzyme buffer two by adding 250 units per milliliter of collagenase.
Type five to the DB solution oxygenate EB one, and warm it up to 37 degrees Celsius. Then perform two 12 minute cycles of digestion in the rotating digestion device with three milliliters of 100%oxygenated EB one at 37 degrees Celsius. Remove the solution by pipette aspiration and discard it after each cycle.
Next, prepare six 15 milliliter tubes for cell collection and 80 milliliters of four degree Celsius craft brew solution for eluding the buffers oxygenate EB two and work it up to 37 degrees Celsius. Subsequently, perform a first 15 minute digestion cycle with three milliliters of 100%oxygenated EB two at 37 degrees Celsius. After the digestion cycle, collect the solution containing the first associated myocytes in a 15 milliliter tube and dilute the cell suspension with 12 milliliters of cold KB solution.
Store the tube flat at room temperature, perform five other 12 minute digestion cycles with three milliliters of EB two at 37 degrees Celsius and collect the myocyte containing buffer at each cycle in a 15 milliliter conical tube. Remember to also dilute the three milliliter collected buffer with 12 milliliters of KB solution at each cycle. At the end, store the six cell containing tubes at room temperature.
In this step, add one milligram per milliliter bovine serum albumin to 20 milliliters of calcium free tyro buffer. Then filter the solution next centrifuge the six myocyte containing conical tubes at 100 cheese for five minutes. To force the myocytes to settle, remove the supernatant and resuspend the cells in each tube with a variable amount of BSA containing TB at room temperature.
Gradually increase the calcium concentration in the cell containing buffer by adding small OTTs of 100 millimolar per liter calcium chloride solution in the first and second steps. Calcium concentration is raised to 50 micro molars per liter and 100 micro molars per liter respectively. The following calcium addition steps are performed every five minutes, and the concentration is raised by 100 micromolar per liter at each step to a final concentration of 0.9 millimolar per liter.
To assess the yield of the isolation procedure, transfer 0.5 milliliters of myocyte containing solution onto the glass bottom chamber of a microscope. Evaluate 15 microscope fields at 10 x objective magnification and calculate the percentage of healthy myocytes such as the rod-shaped cells with clear striations and no significant inclusions. The expected yields should be around 20%to demonstrate human cardiomyocyte functional assessment, including simultaneous recordings of action potentials and intracellular calcium fluxes.
First, prepare the pipette solution for patch clamp experiments in perforated patch configuration. Then add 1.8 millimolar of calcium chloride to calcium free tb. Use the solution for superfusion of cardiomyocytes during patch clamp fluorescence experiments.
Next, transfer one milliliter of cell suspension to a 1.5 milliliter tube and add 10 micromolar per liter flu fort and 10 microliters of power load. Concentrate incubated for 30 minutes at room temperature setting in a horizontal position. Afterwards, set the tube in a vertical position and leave the cells to settle for five minutes.
Then pipette out the supernatant and resuspend the cell pellet in calcium containing tb. Transfer point 25 milliliters of cell suspension to a small temperature controlled microscope mounted recording chamber. Super fused by gravity with a heated micro fuer system at a flow rate of 0.3 milliliters per minute at 37 degrees Celsius.
Using a micro pipette puller, prepare patch clamp pipettes with a tip diameter of three to five micrometers and a resistance of three to 4.5 mega ohm when filled with ps. After that, add amphotericin B to PS at 250 micrograms per milliliter and use it to fill the electrodes, then mount the electrode on the pipette holder. Next, select the Ron shaped cell with clear striations and devoid of inclusions.
Form the giga seal and wait five to 10 minutes until access resistance drops below 20 ohm. Subsequently elicit action potentials in current clamp mode using short pulses at different frequencies of stimulation. During the recording phase, turn on bright field illumination at 492 nanometers and detect Fluor fort fluorescence at 505 to 520 nanometers.
Then acquire the fluorescence and membrane potential signals. This figure shows the alterations of action potentials in ventricular cardiomyocytes from the HCM samples. Here are the representative superimposed action potentials elicited at 0.2 hertz, 0.5 hertz, and one hertz from a control myocyte and an HCM myocyte.
The superimposed action potentials at 0.5 hertz from a control myocyte. In the absence and presence of 10 to seven molar isoproterenol are shown, and here is the representative trace of the membrane potential of a cardiomyocyte from an HCM patient, which displayed several spontaneous depolarizations early after depolarizations. The figure shows the alterations of calcium transient in ventricular cardiomyocytes from the HCM samples.
Here are the representative superimposed calcium transient elicited during stimulation at 0.2 hertz via the patch pipette in a control myocyte and an HCM cell. The kinetics of calcium transients in HCM and control cardiomyocytes at different times are shown, and here are the representative long traces showing intracellular calcium during stimulation at three different frequencies, which highlights the increased diastolic calcium at high pacing rates in the HCM myocyte. This figure shows the additional experimental applications using human ventricular myocytes.
The representative superimposed traces showing L type calcium current recorded at different membrane voltages is shown at the left, and the average L type calcium current peak density from 18 cells isolated from HCM samples at different membrane voltages is shown at the right and shown here is the intracellular calcium trace recorded from a ventricular myocyte during electrical field stimulation at one hertz. While attempting this procedure, it's important to remember to start the procedure shortly after the collection of specimen from the operating room in order to ensure ses Following this procedure to isolate human ventricular cardiomyocytes. Other methods can be performed like lifestyle confocal microscopy or immunochemistry, and this will be crucial to answer fundamental questions such as, for instance, how the subcellular localization of membrane structures and calcium release units is haltered in cardiac diseases After its development.
This technique paved the way in cellular cardiology to study the functional abnormalities in ventricular cardiomyocytes and to test the potential utility of novel therapeutic options. After watching this video, you should have a good understanding of how to isolate viable single cardiomyocytes from human specimens and how to characterize the cell function in different conditions.
