The overall goal of this experiment is to achieve a detailed assessment of the murine atrium with a high temporal and spatial resolution during normal sinus rhythm and atrial arrhythmia induced by electrical stimulation. This method can help answer key questions in the cardiac electrophysiological field, such as how genetic mutations contribute to atrial arrhythmias. The main advantage with this technique is that we can obtain the precise optical mapping in the atria, focusing on the initiation and maintenance mechanism of atrial arrhythmias.
Details on the Langendorff apparatus set up, including the assembly of the perfusion circuit, are provided in the text protocol. Imaging parameters are also provided in the text. To prepare the mouse for cannulation, treat it with heparin and anesthetize it as detailed in the text.
Be certain to confirm the anesthesia with a reflex test before proceeding. To proceed, secure the mouse in a supine position on the operating board. Then, open the abdominal wall below the xiphoid process level using scissors.
Next, make a transverse incision in the diaphragm and cut both sides of the ribs in the medial axillary line without damaging the heart. Then, rapidly excise the heart together with the adjacent vessels and tissues. Next, wash the heart with 10 milliliters of ice-cold PBS and remove the surrounding tissues.
Then, into the ascending aorta, introduce the tip of the 21 gauge blunted needle connected to the perfusion circuit. Under a stereoscope, fix it in position with sutures. Now, perfuse the heart with Tyrode's solution aerated with pure oxygen for 10 minutes.
During the perfusion, continuously monitor the perfusion pressure. Keep it between 80 and 100 millimeters of mercury. During the perfusion, insert thin, polyethylene tube into the superior vena cava and use sutures to hold it in place.
Then, place the heart into the warmed glass chamber. Once in the chamber, puncture the left ventricular cavity with a 24 gauge in-dwelling needle by going through the ventricular apex to avoid damaging the atrium. Then, remove the inner needle, leaving an external cannula in the left ventricle.
Next, introduce an 1-French, custom-made, quadripolar electrode catheter, through the tube in the superior vena cava to stimulate the right atrium. Now, insert a pin electrode into the ventricular apex to continuously record the bipolar electrocardiograms between the pin electrode and canulation needle. Continuously monitor the electrocardiogram and perfusion pressure throughout the entire study.
To stain for single recordings of the membrane voltage, first minimize the ambient lighting. The perfusion solution temperature must be maintained at 37 degrees Celsius. Deliver 10 milliliters of stain over two to five minutes via the perfusion route.
Next, chase out the stain by perfusing Tyrode's solution for five minutes. Be sure to keep the perfusion pressure between 80 and 100 millimeters of mercury. Next, administer one milliliter of the diluted blebbistatin via the perfusion route.
Confirm elimination of heart contraction. Also, make certain that the heart has homogeneously taken up the stain. Before proceeding, set the pacing electrodes and stimulator and find the pacing threshold.
To proceed, carefully put a cover glass on the perfused heart to flatten the atrial surface and prevent motion artifact from vibration of the solution. Confirm that the atrium attaches to the cover glass appropriately. Now, various pacing regiments can be evaluated.
For constant or burst pacing, deliver 99 beats at a pacing interval starting at 150 milliseconds or the longest interval that avoids outpacing the intrinsic rhythm. Then, reduce the pacing interval progressively by five millisecond steps down to 40 milliseconds, or until the interval fails to excite the atrium. For single, extra stimulus pacing, set the pacing cycle lengths of the basic drive or the S1.At least three S1 to S1 intervals should be tested, such as 120, 100 and 80 milliseconds.
Next, set the first extra stimulus, or S2, to negative 10 milliseconds for the basic cycle length, and deliver the S2 after the last pacing stimulus of the basic drive. Gradually, shorten the coupling interval of S2 progressively, by five millisecond steps until the S2 fails to depolarize the atrium. Determine the effective refractory period as the longest S2 interval that fails to depolarize the atrium.
When determining the patient threshold or effective refractory period, it is sometimes difficult to confirm the atrial capture by observing pacing on the electrocardiogram. In such cases, you should check it using optical mapping. To proceed, apply the double and triple extra stimuli pacing to induce atrial tachyarrhythmias according to the text protocol.
Membrane voltage in a Langendorff heart was recorded by monitoring the fluorescence signal from di-4-ANEPPS at 10, 000 frames per second. An activation map was obtained by constant pacing delivered from the right atrium and a detailed conduction pattern in the small murine atria could be analyzed. RH237 and Rhod2AM were used to monitor calcium transience in the left atrium during the same constant pacing protocol.
Sampling at 1, 000 frames per second was fast enough to compare action potentials in the calcium transience. Next, atria tachyarrhythmia was generated in hearts taken from mice 10 days after transverse aortic constriction procedure, that applied a pressure overload to the atrium. Triple extra stimuli pacing from 120 to 80 milliseconds was used to generate the tachyarrhythmia and the propagation of tachyarrhythmia was observed passing through the re-entrance circuit.
Once mastered, this technique can be done in three hours if it is performed properly. While attempting this procedure, it is important to remember to keep the atria free from any damage or stress which could alter the atria's electrophysiological properties.
