Isolating the atrial myocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding atrial electrophysiology at both the cellular and molecular levels. The approach we use effectively yields large numbers of isolated atrial myocytes that can be used to investigate cellular atrial electrophysiology and arrhythmogenesis in a wide range of experimental models and conditions. We have adopted a trunk digestion approach for isolating atrial myocytes.
The advantage of this approach is that it allows the researcher to choose the specific region they wish to investigate. Begin this procedure with preparation of equipment and solutions as described in the text protocol. Lay out the dissecting plate, dissecting tools, a Pasteur pipette, and the fire polished pipettes.
This protocol can be performed on male or female wild type mice, mice carrying genetic mutations, and mouse models of disease. Following mouse euthanasia as described in the text protocol, place the mouse on a paper towel or a cork board and tape the paws down to hold the mouse in place. Wet the chest of the mouse with 70%ethanol.
Remove the fur and skin covering the chest using curved scissors. Next use rat tooth forceps to lift the sternum and then cut the diaphragm along the edge of the ribs. Remove the entire ribcage using curved scissors to expose the heart.
To remove the atrial appendage, gently lift the appendage using fine dissecting forceps and cut it out with spring scissors. Immediately transfer the atrial appendage to a silicon coated dissecting dish containing 20 milliliters of warmed modified Tyrode's pH 7.4 solution. Place one dissecting pin at the top and one pin at the bottom of the opening of the atrial appendage.
Using a Pasteur pipette, flush the atria with the warmed modified Tyrode's pH 7.4 solution to remove the blood. Open the atrial appendage by cutting along its top and bottom edge. Next, pin the corners of the atrial appendage down to create a flat rectangular piece of tissue.
Cut the atrial appendage into approximately eight to 10 equal sized strips using spring scissors and fine forceps. Note that the strips contract once they are cut free from the main piece of tissue. Using the small bore fire polished pipette, transfer the tissue strips into the first tube containing warmed modified Tyrode's pH 6.9 solution.
Wait five minutes. Now, use the medium bore fire polished pipette to transfer the tissue strips to the second round bottomed tube containing modified Tyrode's pH 6.9 solution. To wash the tissue strips, cap the five milliliter round bottomed tube and gently invert the tube three times.
Let the tissue strips settle to the bottom of the tube before transferring the tissue strips to the third tube using the medium bore fire polished pipette. Wash the strips again by inversion. Then, transfer the tissue strips into the enzyme solution using a medium bore fire polished pipette and incubate for 30 minutes.
Swirl the tube every three to five minutes to prevent the tissue strips from adhering together. At the beginning of the enzymatic digestion, tissue strips settle quickly following swirling. At approximately 20 minutes of digestion, the tissue strips begin to float in the enzymatic solution following swirling.
During this time, the atrial tissue strips also change in appearance from pale pink to white as they are digested. After enzymatic digestion, perform three washes using 2.5 milliliters of KB solution in the prepared five milliliter round bottomed tubes. For each wash, gently invert the tube three times before moving the tissue to the next tube using the medium bore fire polished pipette.
Following the final wash, transfer the strips into the 14 milliliter round bottomed tube containing 2.5 milliliters of KB solution. Wait five minutes. Gently triturate the tissue for 7.5 minutes using the wide bore fire polished pipette.
This will mechanically dissociate the tissue strips and yield a cloudy solution filled with individual atrial myocytes. The first of the trituration must be tailored to yield a large number of cells without damage to the cells. Consider factors such as the age of the mice and disease condition.
Tailor the force of trituration to the individual isolation by altering both the frequency and velocity of expelling the tissue strips from the wide bore fire polished pipette. Gentle trituration results in a low cell yield, while harsh trituration will yield many dead cells. Fill the 14 milliliter round bottomed tube containing the triturated tissue strips with KB solution to a final volume of seven to 10 milliliters depending on the desired density of cells for experimental use.
Place this tube at room temperature for one hour. Following this incubation period, cells can be used for a variety of experiments for up to seven hours. Shown here are examples of isolated atrial myocytes from normal mice.
Isolated atrial myocytes are typically on the order of 100 microns in length and 10 microns in width with clear striations. The capacitance of isolated atrial myocytes is typically 40 to 70 picofarads. An example of an atrial myocyte action potential recorded using the perforated patch-clamp technique in current clamp mode is shown.
A representative family of sodium currents was recorded in the whole cell configuration of the patch-clamp technique. These currents were recorded using 50 millisecond voltage clamp steps between negative 100 and positive 10 millivolts from a holding potential of negative 120 millivolts. A summary sodium current voltage relationship is presented here.
A representative family of calcium currents was recorded using 250 millisecond voltage clamp steps between negative 60 and positive 80 millivolts from a holding potential of negative 70 millivolts. A summary calcium current voltage relationship is displayed here. A representative family of potassium currents was recorded from a holding potential of negative 80 millivolts using 500 millisecond voltage clamp steps between negative 120 millivolts and positive 80 millivolts.
Here the summary current voltage relationship is shown for total potassium current. It is important to remember that a successful atrial myocyte isolation depends on both an appropriate enzymatic digestion as well as mechanical dissociation of the cells during trituration. Once isolated, atrial myocytes can be used in patch-clamp experiments for the measurements of action potential morphology and ionic currents, such as sodium currents, calcium currents, and potassium currents.
Investigating changes in atrial myocyte electrophysiology has been essential for determining the cellular and molecular basis of life threatening atrial arrhythmias and for the development and testing of anti-arrhythmic compounds for atrial arrhythmias.
