The overall goal of this procedure is to evaluate sarcoplasmic reticulum calcium reserve and intracellular diastolic calcium removal function in isolated cardiomyocytes. This method can help answer key questions about intracellular calcium handling mechanism under some pathophysiological conditions such as diabetes and heart failure. The main advantage of this technique is the convenience and is less limited by technique and equipment.
To begin this procedure, set up the Langendorff perfusion system. Fill the perfusion system with NT solution, set the temperature at 36.5 degrees Celsius, and eliminate any air bubbles in the tube. Next, open the abdominal cavity under the xiphoid process, lift the xiphoid process and open the chest with a scissor.
Then remove the pericardium, slightly lift the heart with curved forceps, identify the aortic arch, and cut off the heart from the root of the aorta. After that, transfer the heart to a 60 millimeter dish and wash it with NT solution. Hold the aorta with two micro dissecting forceps and mount in onto the Langendorff perfusion cannula.
Then firmly ligate the aorta onto the cannula using surgical sutures. Subsequently perfuse the heart with 30 milliliters of NT solution to recover circulation of the coronary arteries. Then switch the perfusate to 30 milliliters of calcium-free Tyrode solution to stop the heartbeat.
Afterward, switch the perfusate to collagenase A isolation solution for enzyme digestion. After digestion for 20 to 25 minutes, quickly change the perfusion solution to the calcium-free Tyrode solution to stop further digestion. Then hold the heart with forceps, cut it off from the cannula, and place the heart in a 35 millimeter dish containing KB solution.
Following that, find the left ventricular area. Cut off the atrium, right ventricle, and atrioventricular junction area. Then transfer the left ventricle to a new 35 millimeter dish with KB solution and mince the tissue into small pieces.
Gently pipette the pieces with a filtered plastic dropper. Next, filter the cells with a 150 micron aperture stainless steel filter and transfer them to a 15 milliliter centrifuge tube. Following that, centrifuge the sample at 150 times g for 30 seconds and discard the supernatant.
Resuspend the myocytes in 10 milliliters of KB solution and let them settle for six minutes. Afterward, discard the supernatant and resuspend the pellet in 10 milliliters of KB solution for 20 minutes. After settling in KB solution for 20 minutes, discard the supernatant and resuspend the myocytes with calcium reintroduction solution A.Repeat the above procedure with calcium introduction solution B.Then repeat the above procedure with NT solution to purify the available myocytes.
Store the isolated LV myocytes in this solution and study them within four to six hours. Then add NT solution and 10 millimolar caffeine solution to each column of the pencil. Evacuate any air bubbles in the tubes to avoid air blockage.
Now, connect the inflow tube with the NT solution for chamber perfusion. For needle perfusion of the target myocyte, connect the multi-barrel manifold tip to the valve controlled perfusion system. Afterward, count the drip number from the micron tip of the pencil for 10 seconds and manually adjust the flow regulator to keep the flow speed at an approximate velocity of three drips per 10 seconds.
In this procedure, add the fura-2-acetoxymethyl stock into one milliliter of the myocyte suspension to bring the final concentration to two micromolar. Keep them in the dark for 20 minutes at room temperature. After that, discard the supernatant and resuspend the cardiomyocytes with NT solution twice.
Next, turn off the light and keep the cells in the dark. Place the myocytes in the perfusion chamber for 15 minutes before perfusing them with NT solution. Pace the myocytes with one hertz field stimulation for five minutes using a stimulator.
Now, select a myocyte with good shape and stable stimulated twitch under the 10X microscopic objective lens. Click File New File to create a new recording file. Next, change the microscopic magnification to 40X and rotate the CCD camera orientation to keep the myocyte in a horizontal position.
Then frame the single myocyte by adjusting the cell framing adapter and ensure that the background is clear. Subsequently expose the myocytes to the light emitted by a Xenon lamp with 340 or 380 nanometer wavelength excitation filters and image the myocytes through a 40X objective. Detect fluorescent signal at 510 nanometers.
Click the Start button to synchronously record fluorescence and sarcomere length using the dual excitation fluorescence photomultiplier system. Note the sarcomere length changes of the myocytes and record using the soft edge module simultaneously. In this procedure, interlink the aux out port in the stimulator with the analog import on the valve control system by a BNC cable.
Next, preset the parameters in the valve control software, click the download button to download the sequence loaded in the program, and click the trigger button to enable the trigger function. Then preset the stimulator to sequence mode and set the parameters. Afterward, set the stimulator at S1 step to pace the LV myocytes at one hertz.
Start needle perfusion at the speed of 1.5 milliliters per minute with NT solution. Select a target myocyte under the low power microscopic view and make sure it can be reached by the microtip of the pencil. Following that, change the microscope magnification to 400X.
Rotate the CCD camera orientation to keep the myocyte in the horizontal position. Then adjust the rectangular aperture under the cell framing adapter to a suitable window that fills with the myocytes. Next, adjust the position of the pencil fixed on the micro manipulator and carefully place the microtip at the distance of the radius of vision field to the target myocyte.
Then adjust the needle stream range to mostly cover the target myocyte and make sure that the myocyte cannot be swept away by the needle flow. After 60 seconds basic pacing, roll the stimulator cursor to the D2 step. The rest of protocol will be executed automatically by stimulator and valve commander.
Click the Pause button to pause the file recording and move the microscopic view to a nearby blank area. Then click the Record button to resume recording for seconds and record numerical 340 and 380 nanometer intensity values for background correction. After that, open the Operation Constant dialogue box and fill the values into the background form.
The software can correct fura-2 ratio values by subtracting the background. Shown here is an illustration of the protocol for switching perfusion valves and pacing system automatically. This figure shows the statistic values of sarcoplasmic reticulum calcium reserve and fractional calcium release for one hertz stimulated twitches in the diabetic and Sprague Dawley groups.
The diabetic group showed significant decreased SR calcium reserve and SR fractional calcium release than the Sprague Dawley group. Here, the software panel shows a manually mono exponential curve fitting for the decay time constant of caffeine-induced calcium pulse. These bar graphs compare the Tau-caffeine and Tau-1Hz/Tau-caffeine between the diabetic and Sprague Dawley groups.
The diabetic group showed significant increased values of Tau-caffeine and Tau-1Hz/Tau-caffeine than the Sprague Dawley group. This procedure provides a feasible and a convenient tool to explore intracellular calcium handling mechanism of cardiac dysfunction. While attempting this procedure, there are some key points that should be noted such as the stable adherence of myocytes to the dish, wrap the channel switch, while control the flow velocity of the needle perfusion and the appropriate distance between the needle tip and the target myocyte.
