The goals of this protocol are to use LOR perfusion to isolate cardiac myocytes from adult mouse hearts with known genetics and to analyze contractility and calcium transients of the myocytes. After a heparin injection, the heart is removed from an anesthetized mouse. It is quickly mounted via the aorta to a cannula attached to a syringe of perfusion buffer.
The aorta is secured. A suture and buffer is slowly perfused to clear the coronary arteries. The cannula is then mounted onto a LOR apparatus with flowing perfusion buffer.
The heart is digested with a calcium free solution of collagenase and protease until it is palpably soft. The left ventricle is placed in the calcium free buffer and dispersed with forceps and gentle pipetting. After the solution is filtered through mesh cells are gradually introduced to higher calcium concentrations to remove dead cells and ensure myocytes are calcium tolerant.
Healthy cells are rod shaped while dead cells lack this form. Myocytes are then loaded with RA two A radiometric calcium binding dye, and placed on the stage of the microscope. For analysis, cells are electrically paced while contractility is measured using sarcomere length and edge detection and calcium Transients are measured by analyzing the spectral shift of the RA two loaded cells.
Dilated cardiomyopathy is caused by a variety of mutations. However, understanding the mechanism behind this heart disease can be difficult. Isolating cardiomyocytes allows us to analyze the contractility and calcium transients of the Contractal cell of the heart, Calcium Free, And 1.2 millimolar.
Calcium tyro solutions can be prepared in advance on the day of the isolation. Prepare the calcium free profusion buffer enzyme digestion, buffer, and transfer buffers A and B per the written protocol. Filtering all solutions through a 0.2 micron filter before use.
Make transfer buffers one through four with increasing calcium concentrations by mixing buffers A and B according to the written protocol. Set the flow rate of a LOR apparatus to three milliliters per minute and set the circulating water bath temperature so that the outflow from the cannula tip is about 37 degrees. Run 70%ethanol through the system for 15 minutes, followed by a hundred milliliters of water, ensuring all bubbles are removed from the system.
Before your first isolation, create a cannula by attaching a short piece of PE 50 tubing to the tip of a 20 3G lure stub adapter, and heat the tip of the tubing to create a small lip. Attach the cannula to a small lu lock syringe filled with perfusion buffer. Ensure the cannula is filled with buffer.
Inject a mouse with 200 heparin via IP injection. Begin running profusion buffer through the L endorf so that it is fully run through the system before the heart is attached. After five minutes, anesthetize the mouse with isof fluorine.
Ensure the mouse is fully anesthetized to firm bilateral toe pinch. Open the chest by cutting below the sternum and up either side of the rib cage quickly excise the heart being careful to leave as much aorta as possible attached to the heart. Immediately place the heart in ice.
Cold profusion buffer, working as quickly as possible Under a dissection microscope, remove any extra tissue. Work carefully to remove excess fat without damaging the underlying Aorta. Trim the aorta to immediately below the first branches.
Using two Pair of fine forceps, carefully grasp the edge of the aorta and pull gently over the tip of the cannula. Ensure the tip of the cannula does not pass through the aortic valve Into the left ventricle. Secure the aorta with a small loop of 5.0 suture.
Gently push a small amount of perfusion buffer through the cannula, watching for the coronary arteries to clear of blood. Remove the cannula from the syringe and attach to the L endorf apparatus with flowing perfusion.Buffer. Careful not to introduce any bubbles.
Allow the perfusion buffer to flow through the heart for two to three minutes. Perfuse eight should go from bloody to clear switch flow to digestion, buffer, and digest for seven to 10 minutes. After the buffer has filled the lang endorf flow can be recycled once the heart is palpably.
Flacid, remove from cannula and place in a P 60 dish with Buffer a. Remove The atria, great vessels and right ventricle and transfer to a second dish of buffer a. Using forceps.
Gently separate the left ventricle into small pieces. If the heart is adequately digested, tissue should separate easily pipette gently several times with a sterile five mil transfer pipette To further disperse the cells. Use a sterile past pipette to transfer solution containing cells and tissue through a 250 micron nylon mesh Filter into a 50 milliliter conical tube.
Allow the cells to pellet for approximately 10 minutes. Live cells will settle to the bottom in the pellet and dead cells will float. Transfer the pellet to the 0.06 millimolar calcium transfer buffer.
Allow to sit for 10 Minutes, Aspirate the supernatant containing the dead cells, and transfer the pellet to the 0.24 millimolar calcium solution. Repeat pelleting aspiration and transfer through increasing calcium solutions until the cells have pelleted in the 1.2 millimolar calcium transfer buffer. At this point, the pellet will contain many healthy rod-shaped cells, not spontaneously contracting to load cells with fewer or two dye.
Transfer one mil of resuspended cells into a one mil eend orph tube. Add one microliter of one millimolar fira two in anhydrous DMSO. Mix by gently inverting the tube.
Allow the cells to pellet for 15 to 20 minutes in the dark after the cells have pelleted wash by aspirating the supernatant and adding 1.2 millimolar calcium tyro solution. Mix and allow cells to settle for five to 10 more minutes. Then repeat the wash.
Set up the inverted microscope for data collection. First place a glass cover slip on a stage insert designed for profusion aspiration, and for electrical pacing of cells. If using an oil objective, place a drop of oil on the 40 x lens and place the stage insert on the microscope.
Attach the electrodes. Set up a gravity driven heated profusion of 1.2 millimolar calcium tyro solution and attach aspiration to vacuum. Turn on the microscope and cell pacer system.
Open the ion wizard software. Load A few drops of few or two loaded cells onto the stage while blocking the perfusion flow. To allow healthy cells to settle, restore profusion, many of the remaining dead cells will be washed away.
Use the eyepiece to locate a healthy rod-shaped cell that contracts upon pacing at five to 20 volts spontaneously, contracting cells should be avoided due to membrane leakage. Change the visual output to the camera. Adjust the rotation of the lens so the cell is visible in the window.
Adjust the aperture so that most of the background is removed. Place the sarcomere measurement marker over an area of the cell with uniform sarcomeres. Change the size of the box if necessary to ensure the marker is not covering different populations of, if desired.
Align the edge detection Bars as well. Begin pacing the cells 10 to 15 seconds before starting the experiment. Start recording.
Analyze sarcomere shortening and URA two ratio traces with the ion wizard software. Possible comparisons between wild type and knockout cardiomyocytes include fractional shortening time to peak shortening time to 50%relaxation for contractility analysis, as well as baseline peak time to peak and time to 50%baseline fear or two ratios to assess calcium flux. The cells produced with this protocol are ideal for many other experiments and procedures, including staining for subcellular components and short-term culture.
Now that you've seen this video, you should be able to isolate cardiomyocytes from wild type and knockout mice and to analyze the contractility and calcium transients of these cells. Good luck with your experiment.
