The overall goal of this technique is to obtain viable ventricular heart slices, which are suitable for electrophysiological recordings. This method can help answer key questions in the field of cardiac electrophysiology on the tissue level, such as on electrical integration of transplanted cardiomyocytes or effects of cardioactive drugs. The main advantage of this technique is that viable ventricular tissue slices have a preserved, in vivo-like tissue structure, and it enables direct measurements in all regions of the heart.
Though this method can provide insight into murine heart electrophysiology, it can also be applied to other animals, like rabbit or guinea pig. To begin this procedure, first wipe the skin of the animal with 70%ethanol. After opening the thorax, carefully dissect the heart's pericardium, using small scissors and forceps.
Insert a cannula into the left ventricle and perfuse the heart in situ with ice cold Tyrode solution without calcium, until the remaining blood is removed. Then, gently resect the heart and transfer it to the ice cold Tyrode solution without calcium. Separate the atria from the ventricles with a scalpel or scissors.
Next, place the ventricles with the apex, facing upwards, in the agarose mold. Subsequently, place the pin in the middle of the mold and in the left ventricular chamber. Fill the mold with 4%low-melt agarose at 37 degrees Celsius until the heart is completely covered.
Then, place the mold on ice for faster agarose hardening to prevent the tissue from floating. After one minute, remove the agarose block, containing the ventricles, from the mold with a scalpel. After that, turn the block upside down and fill the ventricular chambers and the gap on the backside of the agarose block with 4%low-melt agarose using a syringe.
Then, trim the agarose block with a scalpel to achieve a flat bottom and upright position of the cardiac apex. In this procedure, fix the block on the specimen holder of the microtome with a drop of cyanoacrylate glue, with the cardiac apex facing upward. Next, place the specimen holder into the inner specimen chamber of the microtome, which is filled with ice cold Tyrode without calcium.
Ensure that the agarose block is completely covered with Tyrode solution. Prepare the slices at a thickness of 150 to 400 micrometers, depending on further application. Keep the vibration frequency of the blade, at 60 to 70 Hertz, and move the blade forward, as slowly as possible.
Afterward, use a fine brush to carefully remove the remaining agarose from the slices. Then, gently transfer the slices with a Pasteur pipette into the Tyrode solution, with 0.9 millimolar calcium, aerated with 100%oxygen. Store them for at least 30 minutes on ice to recover from the slicing procedure.
Placing the slices on a net helps keep them flat. Afterwards, use a filter paper and a second net to transfer the slices in DMEM at 37 degrees Celsius, bubbled with carbogen, to wash out BDM before further use. For preheating, switch on all the electric devices 30 minutes before the recordings.
Place a three centimeter cell culture dish with the heating plate on the inverted microscope. Then, place the custom-made ring electrode in the dish, and connect the grounding wires of the preamplifier and the stimulation electrode. After that, connect the flexible tubes of the perfusion system to the dish.
Fill the reservoir of the perfusion system with DMEM and aerate it with carbogen. Then, switch on the perfusion pump and set the perfusion rate to two to three milliliters per minute. Adjust the temperature of DMEM in the dish to 37 degrees Celsius, by regulating the flow heater and the heating plate.
Now, place a ventricular slice into the DMEM-filled dish. With the inverted microscope, check the structural integrity and viability of the tissue. Then, fill a recording glass electrode with three molar potassium chloride, and a stimulation electrode with DMEM.
Next, place the recording electrode in the stimulation electrode on the electrode holders. Subsequently, place the stimulation electrode carefully on the slice. Switch on the electric stimulator and start with a stimulation frequency of one to two Hertz.
Then, move the recording electrode with a micromanipulator to the intended recording position. Slowly lower the recording electrode until the tip touches the tissue. Once the recording detects the contact, apply a short rectangular electric pulse through the recording electrode to penetrate the cell membrane.
Next, carefully reposition the recording electrode until a stable signal is ensured. Following that, begin the recording of action potentials. To assess integration and maturation, cardiomyocytes, derived from iPSCM expressing eGFP, were transplanted into the healthy hearts of adult mice.
Shown here is a ventricular heart slice, containing the transplanted iPSCM, which was focally stimulated by a unipolar electrode, placed in the host tissue. Intracellular action potentials were recorded with sharp glass microelectrodes filled with three molar potassium chloride and eGFP-positive transplanted iPSCM, and neighboring host tissue within the slices. Here are the action potential recordings, in the healthy host tissue and the transplanted iPSCM.
The slice was focally stimulated with a stimulation electrode placed in the host tissue, at around two Hertz and five Hertz. Once mastered, this technique can be done in one to two hours, if it is performed properly. While attempting this procedure, it is important to remember to keep the forward speed of the microtome blade as slow as possible.
Following this procedure, other methods like first measurements can be performed in order to answer additional questions. After its development, this technique paved the way for researchers in the field of cell therapy to explore electric and integration, and maturation of transplanted cardiomyocytes in recipient hearts. After watching this video, you should have a good understanding of how to prepare viable ventricular short axis slices, using a microtome with a vibrating blade.
These slices are particularly suitable for electrophysiological recordings sharp glass electrodes.
