The procedure begins with taking the heart out of adult rats. The heart is digested and single cardiomyocytes are isolated. The isolated cells are cultured.
Adenoviral gene transfer leads to the expression of fluorescent fusion proteins or genetically encoded biosensors cardiomyocytes expressing the reporter protein are visualized by confocal microscopy and further analyzed. Hi, I am Kain from the Institute for Molecular Cell Biology in the medical faculty at Sland University. I'm Lars also from the Institute for Molecular Cell Biology, And I'm uncle from the same lab.
Today we will show you a procedure for isolation of cardiac myocytes transduction with fluorescent fusion proteins or genetically encoded biosensors and the preceding confocal imaging procedure. We use this procedure in our lab to study cardiac physiology, pathophysiology, and pharmacology at the cellular level. So let's get started.
To begin this procedure, prepare a LOR perfusion setup, prepare and fill in all the solutions and preheat the devices to 37 degrees Celsius. Make sure all solutions are saturated with oxygen and all air bubbles are removed from the tubing. Use six to 12 week old adult male wistar rats weighing approximately 250 grams and pre sedate them by carbon dioxide inhalation to ease animal handling.
Anesthesia is performed by an intraperitoneal injection of 170 microliters of anesthesia solution per 100 grams of body weight. Additionally, inject citrate solution at two microliters per gram body weight to avoid blood coagulation during the surgery. When the rat is anesthetized, fix it on a surgery board and disinfect the upper part of the body with 70%ethanol.
Sacrifice the animal by cutting through the carotid, then use scissors to open the thorax. Now that the heart and the aorta are accessible, remove the peric card. Using a 26 gauge needle, inject five milliliters of ice cold solution a plus into both ventricles in order to arrest the heart and wash out the blood.
Next, grab the aorta with a clamp and truncate at the aortic arch just where the first branching occurs with the aorta. Now truncated, remove the heart from the chest and use forceps and a clamp to attach the aorta to the cannula of the perfusion setup. Be careful not to ruminate the aortic valves.
The heart needs to be fixed by a ligature around the aorta. Cannula, assembly, and open vessels need to be ligated as well. Now one can increase the retrograde perfusion of the heart with oxygen saturated solution, a plus at room temperature for 10 minutes at a pressure of approximately 70 millibar.
Make sure no air bubbles enter the aorta at the end of 10 minutes. Switch the perfusion solution to a liase solution at 37 degrees Celsius for approximately 25 minutes at a rate of four milliliters per minute, using a peristaltic pump at the end of the perfusion, the heart should look marbled white. The digestion time may need adapting to the size of the heart.
After digesting with the liase solution, remove the heart from the perfusion setup and place it in a Petri dish containing solution A.At a temperature of 37 degrees Celsius, cut off the remaining vessels and atria. Then divide the ventricles into approximately six pieces. Transfer the cut ventricle pieces into a 100 milliliter erlenmeyer flask containing 20 milliliters of solution A at a temperature of 37 degrees Celsius and agitates lightly in a 37 degrees Celsius water bath shaker for five minutes.
At the end of five minutes, the tissue pieces remain mostly at the bottom of the flask and the supernatant appears slightly opaque. Discard it and repeat the gentle agitation. Next, increase the extracellular calcium concentration by adding 20 milliliters of solution B one half at 37 degrees Celsius to the cells and tissue remaining in the erlenmeyer flask.
Gently shake the erlenmeyer in a 37 degrees Celsius water bath shaker for an additional five minutes and discard the supernatant again. Add 20 milliliters of solution B one. Half the solution now contains cells which are viable.
Transfer a drop of the cells to a Petri dish and examine using an inverted cell culture microscope. If the cells show high spontaneous contractions, one will need to wait until the cells come to rest. Once more, the supernatant is discarded and 20 milliliters of solution B one half is added to manually disrupt the tissue, cut off the tip of a plastic past your pipette so that the tissue pieces can easily pass through the opening flame.
The cut tip so that the sharp edges are softened by melting, proceed to gently and carefully iterate the tissue, which starts to fall apart and release the cardiomyocytes. The supernatant now contains the cells, decant it and dute with solution B to reach a cell density of approximately 5, 000 cells per milliliter with the primary cardiomyocytes isolated, proceed to adenovirus, transduction, and imaging cells should be rod shaped, typically 100 micrometers long and about 20 micrometers wide. They show a distinct transversal cross trition indicating the so-called sarcomere length, which should be 1.8 micrometers in the resting state.
Adenovirus particles carrying the gene of interest were produced in Qbi HEK 2 93 A.Cells as described in the accompanying written protocol virus passage two or higher is used to transduce the heart cells start culting the primary heart cells by placing sterile cover slips. In standard 12, well plates equally distribute 20 microliters of ECM solution on each cover slip and discard. The remainder then allow to dry for a minimum of one hour at 37 degrees Celsius.
When the ECM coating on the cover slips is dry, fill each well with one milliliter of medium, 1 99 with added antibiotics and ITS. Now that the cell culture plates are ready. At 200 to 400 microliters of isolated cardiomyocytes to each well and proceed to culture.
The cells at 37 degrees Celsius in a 5%carbon dioxide incubator allow the cells to sediment for one hour and then change the medium to M1 99 again. Immediately after changing the medium, an adequate amount of virus containing solution is gently mixed with the cell supernatant. 24 hours after transfection, the fluorescent fusion protein expression is detectable and one can proceed to imaging.
For confocal imaging, we use the VT infinity, which is a killer beam array scanner. In addition to high spatial resolution, this instrument allows high temporal resolution at up to 200 frames per second, which can be used in calcium imaging. Have the microscope and peripheral equipment ready and transfer the cover slips holding the cells into an experimental chamber, followed by the addition of one milliliter T road solution.
Focusing and selection of cells is done using white light or epi fluorescent illumination, then proceed to data acquisition. Here we take a Z stack of a cell expressing a fluorescent fusion protein with a targeting sequence to the Golgi apparatus. Finally, analyze the image by 3D reconstruction using a dedicated analysis software IUs by bit.
Plaine is used here. Shown here is the 3D visualization of the Golgi apparatus in the isolated cardiomyocytes. Since the microscopy is performed on living cells, there are no fixation artifacts.
Subcellular structures can be followed upon physiological or pathophysiological stimulation, such as hormonal treatment under otherwise controlled conditions. When expressing genetically encoded biosensors instead of fusion proteins, it is possible to follow functions and even biochemical reactions of the cells. Examples are calcium sensors or phosphorylation sensors.
We've just shown you how to visualize subcellular structures in living cardiac myocytes. The same can be used for a broad set of approaches, including functional visualization. When doing this procedure, it's important to remember to respect the sacrifice, the animal.
So that's it. Thanks for watching and good luck with your experiments.
