Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

This protocol allows the user to achieve efficient transgene expression in the ventricular myocardium, facilitating gene expression studies to examine cardiac arrhythmias and function in the ventricular tissue. The technique for viral transgene expression is minimally invasive for the animal, causing less damage and reduced recovery time than conventional approaches that involve a thoracotomy. The technique for inducing arrhythmias in small rodent hearts is useful for assessing the susceptibility to arrhythmias in small animal models of heart disease.

Demonstrating the procedure will be Doctor Alice Lu, a post-doctoral fellow from the lab. To begin, sterilize the left lower chest region of the anesthetized mouse with alternating rounds of an iodine-based or chlorhexidine-based scrub, in alcohol, three times in a circular motion. Under ultrasound imaging guidance, insert the needle of the syringe containing the virus into the animal's chest.

Approach the needle tip into the left ventricular front free wall and slowly inject 10 to 15 microliters of the virus. Verify successful injection in ultrasound images by the enhanced brightness near the tip of the needle. Withdraw the needle from the heart and insert into other regions of the left ventricle for a second and third injection of the same amount of virus.

To perform Langendorff perfusion of the mouse heart, place the cannulated heart in a silicone-elastomer-coated, 10-centimeter plastic dish, with the left ventricle facing up. Cannulate the aorta of the heart with a blunt needle connected to a modified Langendorff profusion system in the constant flow rate mode. Perfuse the heart with oxygen-bubbled Tyrode solution at 37 degrees Celsius.

At the beginning of perfusion, verify the correct aortic cannulation by observing the washout of blood from the heart during the first two to three heartbeats and changing the heart color from red to pale. Adjust the flow rate to keep the perfusion pressure at 70 to 80 millimeters of mercury. After confirmation, place the electrodes of a small animal ECG system around the heart by inserting them into the silicone-elastomer coating in the dish.

Then, record the ECG using compatible software. Next, perform adrenergic receptor stimulation and perfuse the heart with isoproterenol and Tyrode solution. After 10 minutes, perform programmed electrical stimulation to induce ventricular tachyarrhythmias by stimulating the heart at the apex with two platinum electrodes connected to an electrical stimulator.

Start the stimulation procedure with initial 10 consecutive stimuli, S1, followed by an extra stimulus, S2, with an initial interval of 80 milliseconds. Then, repeatedly reduce the S2 interval by two milliseconds each time until the heartbeat can no longer be captured or the heart's effective refractory period, or ERP, is reached. Monitor any induced ventricular tachyarrhythmias, including ventricular tachycardia and fibrillation, by ECG.

If no arrhythmias are induced. add another extra stimulus, S3, after S2, with the same parameters until the ERP is reached. If ventricular tachyarrhythmias are still not induced, stop the electrical stimulation and consider the heart non-inducible.

During the programmed electrical stimulation, the successful pacing of the heart is verified by the one-to-one capture of the heartbeat during the consecutive S1 stimuli and the prolonged QRS complex during S1 pacing. These combined adrenergic and electrical stimulations induced no ventricular tachyarrhythmias in healthy, sham-operated wild-type mouse hearts, or in control adenovirus-GFP-injected rat hearts. In contrast, the same protocol induced ventricular tachyarrhythmias in 77%of wild-type mouse hearts after myocardial infarction and in three out of four rats after intramyocardial injection of Ad-Wnt3a.

While inserting the needle, ensure that the needle tip is embedded within the free wall and not inside the ventricle. Also, securing and cannulating the aorta of the heart is the most critical step. Following these procedures, hearts can be used for standard histological studies or molecular biology assays.

Alternatively, live cells can be isolated in order to perform single-cell electrophysiology studies.