Cardiovascular malfunction is the leading cause of death worldwide. The isolated heart model that we present here can serve as an experimental window into human cardiac dynamics. This approach combines a classical electrophysiology study with the simultaneous optical mapping of transmembrane voltage and intercellular calcium to evaluate the state of the heart.
This methodology can be applied to disease modeling, pharmacology, and toxicology studies. For example, one of our areas of focus is characterizing the effects of glass sizers on cardiac electrophysiology. Although this methodology is more technically challenging than a protocol that utilizes a smaller animal model, overall, the protocol is relatively straightforward once all the pieces are in place.
It's difficult to describe all of the components of an imaging and perfusion system by text, so visual demonstration of this process will aid in reproducing a successful preparation. After isolating the heart with the ascending aorta intact, plunge the excised organ into ice-cold cardioplegia and use a pair of hemostats to grip the wall of the aorta. Slip the vessel onto a ribbed cannula attached to tubing connected to one liter of ice-cold cardioplegia medium suspended approximately 95 centimeters above the heart.
Allow the fluid to fill the aorta until the vessel overflows to prevent any bubbles from entering the vasculature and use umbilical tape to secure the aorta to the cannula. Tie the hemostats to bear the weight of the heart as it hangs from the cannula to further secure the tissue and allow the cold medium to retrograde perfuse the heart at a constant pressure of 70 millimeters of mercury by gravity. Transfer the hear to the 37 degrees Celsius Langendorff system without introducing air into the cannula.
And allow the normal sinus rhythm to flush the vasculature of any remaining blood and cardioplegia. In the event of shockable arrhythmias, place external paddles at the apex and base of the heart to defibrillate the organ, delivering a single shock at five joules and increasing in five joule increments until 50 joules, cardioversion, or unshockable rhythm is achieved. Then, flush the heart with at least one liter of modified Krebs-Henseleit medium without recirculation to remove any residual blood and cardioplegia.
When the medium runs clear through the heart, close the circulating loop to recirculate the perfusate. To record a standard lead to ECG throughout the course of study, attach a 29-gauge needle electrode to the ventricular epicardium near the apex and attach another electrode in the right atrium. Connect the positive and negative inputs of a differential bioamplifier to the apex and right atrium, respectively, and attach one bipolar stimulus electrode on the right atrium and a second bipolar stimulus electrode to the lateral left ventricle for pacing purposes.
Pace the heart using an electrophysiology stimulator, with the initial current set to twice the diastolic threshold and a one-millisecond pulse width. To identify the pacing threshold, apply a series of one to two-milliamp stimulus impulses with a one-millisecond pulse width at defined pacing cycle lengths to ensure a consistent stimulus response. Perform extra stimulus pacing using either an S1, S1 or S1, S2 pacing train.
Decrease the S2 pacing cycle length stepwise by 10 milliseconds until pacing fails to capture. Then, step up to the penultimate pacing cycle length and decrease the cycle lengths in one-millisecond intervals to determine the most precise pacing cycle length before the loss of capture. To establish the ventricular effective refractory period, use the stimulus electrode on the lateral left ventricle to identify the shortest S1, S2 interval at which the S2 premature beat initiates ventricular depolarization.
To define the Wenckebach cycle length, use the stimulus electrode on the right atrium to find the shortest S1, S1 interval at which a one-to-one atrioventricular conduction propagates via the normal conduction pathway. To define the sinus node recovery time, use the stimulus electrode on the right atrium to apply an S1, S1 pacing train and measure the time delay between the last impulse in the pacing train and the recovery of spontaneous sinoatrial node mediated activity. To establish the atrioventricular node effective refractory period, use the stimulus electrode on the right atrium.
Find the shortest S1, S2 coupling interval at which the premature atrial stimulation is followed by a Hisbundle potential that elicits a QRS complex, signifying ventricular depolarization. For optical mapping of the transmembrane voltage and intracellular calcium, first slowly add up to five milliliters of freshly prepared voltage dye proximal to the aortic cannula followed by the slow addition of freshly prepared calcium dye. Next, position the imaging hardware to focus on an appropriate field of view and connect the camera to a workstation.
Acquire images using the selected software with an exposure time of 5 to two milliseconds. And perform an image alignment with the aid of software that can split and overlay the desired regions and display a grayscale subtraction or pseudo-color addition to any highlight misalignments. With the ambient lighting minimized, test the LED lights to ensure a uniform and maximum epicardial illumination, as determined by the sensor well depth.
Then, image the myocardium during the sinus rhythm, ventricular fibrillation, or dynamic pacing using the stimulation electrode positioned on the left ventricle, beginning with a pacing cycle length of 350 milliseconds and decreasing by 10 to 50 milliseconds to generate restitution curves. To confirm the acquisition of an optical signal of quality throughout the experiment, open a video file, select a region of interest, and plot the mean fluorescence over time. At the end of the experiment, remove the heart from the system and drain the perfusate.
Then, rinse the system tubing and chambers with purified water. For routine maintenance, periodically rinse the system with detergent solution as needed. In these representative studies, the procedure was performed on intact models, as demonstrated, that ranged in size from 2.5 to 10.5 kilograms of body weight and 18 to 137 grams of heart weight.
After transferring the isolated heart to a Langendorff system, the heart rate stabilizes to about 70 beats per minute within approximately 10 minutes of defibrillation and remains constant throughout the duration of study. An average flow rate of approximately 184 milliliters per minute is typically measured. It slows to 70 milliliters per minute after perfusing with warmed medium containing a mechanical uncoupler.
Lead two ECGs can be recorded throughout the duration of the study during sinus rhythm or in response to external pacing to quantify the electrophysiological parameters. Optical mapping experiments can also be performed during the sinus rhythm and spontaneous ventricular fibrillation. Representative images of a dye-loaded heart can be obtained with the corresponding optical action potentials.
And calcium transients can be collected from two regions of interest on the epicardial surface. In addition, dynamic epicardial pacing can be used during the optical mapping experiments to normalize any slight differences in the intrinsic heart rates. The raw signals can be used to depict the action potential calcium transient coupling time, the activation and duration time, and the electrical and calcium restitution.
It is important to avoid allowing bubbles to enter the aorta, to minimize the time of transfer from the animal to the system, and to support the larger heart. Electrophysiology and optical signal data can be analyzed post-acquisition. Additionally, the heart may be preserved after the study for histology, immunostaining, or gene expression analysis.
We're using this technique to characterize juvenile cardiac development and to examine the impact of environmental exposures on cardiac physiology. The mechanical uncouplers used to minimize motion and the anti-foam chemicals used to address albumin frothing are both toxic, so using the appropriate personal protective equipment is absolutely essential.
