Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Eighth Edition). All methods and protocols used in these studies have been approved by the Institutional Animal Care and Use Protocol Committee at Children’s National Hospital following the Guidelines for Care and Use of Laboratory Animals published by NIH. All animals used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.

1. Preparation

1. Prepare 6 L of modified Krebs-Henseleit solution¹⁶ (mM: 118.0 NaCl, 3.3 KCl, 1.2 MgSO₄, 24.0 NaHCO₃, 1.2 KH₂PO₄, 10.0 glucose, 2.0 sodium pyruvate, 2% albumin, 2.0 CaCl₂). Add CaCl₂ on the day of the experiment, since over time, in the presence of phosphates, calcium chloride will terminally precipitate out of solution as calcium phosphate.
2. Adjust the pH to 7.4 after sterile filtering (pore size: 0.22 µm). Check the solution osmolality to ensure a range of 275−310 mOsm/kg. Cool 1 L on ice for use immediately after the heart is excised. Warm 3 L in a water bath to approximately 37 °C before bubbling with carbogen (95% O₂, 5% CO₂).
   NOTE: Warming minimizes bubbles and potential embolism, since cold liquid has an increased capacity of gas to dissolve; therefore, as the modified Krebs-Henseleit media passes through the perfusion system and warms, the gas will be released as bubbles.
3. Prepare 2 L of cardioplegia (modified del Nido cardioplegia solution, Table 1). Freeze enough cardioplegia in an ice cube tray to fill a 500 mL beaker.
4. Turn on the circulating water baths set to 42 °C. Turn on the pumps to circulate the perfusate in a closed hydronic heating loop (for a full list of materials, see Table of Materials and Figure 1).
   NOTE: A heated circulating water bath is used to warm the water-jacketed tubes and heat exchangers.
5. Clean tubing circuits and chambers by running 2 L of a 1% solution of universal detergent in water through the system. Rinse all tubing circuits and chambers of the Langendorff system with >4 L of purified water. Run pumps until all water has been removed from the system.
6. Add a synthetic membrane filter in line with the perfusion pumps (polypropylene filter, pore size >5 µm). Gas a microfiber oxygenator (hemofilter) with 95% O₂ and 5% CO₂ at 80 kPa.
   NOTE: When using albumin, there is often frothing associated with oxygenation and/or the pumping activity through the tubing circuit. An anti-foam compound (antifoam Y30 emulsion) can be added dropwise periodically (~every 30 min) to quench it as it occurs.
7. Check a two-point calibration (0 and 60 mmHg) for the pressure sensor located above the aorta or in the bubble trap; calibrate as needed.
8. Immediately before heart excision, pour the media into the Langendorff loop perfusion system. Ensure that perfusate passes through microfiber oxygenators (hemofilters) gassed with oxygenated perfusate, which then flows through heat exchangers to maintain a media perfusate temperature of 37 °C at the aorta.
9. Set the circulating water bath to a few degrees higher than 37 °C, such as 42 °C, to account for heat loss during exchange and throughout the system. Monitor the circulating perfusate temperature with thermocouples.

2. Heart Excision and Langendorff-perfusion

1. Sedate the pig with an intramuscular (I.M.) injection of ketamine (20 mg/kg) and xylazine (2 mg/kg) and intubate with an endotracheal tube. For induction, administer an intravenous (I.V.) bolus injection of fentanyl (50 μg/kg) and rocuronium (1 mg/kg). Maintain anesthesia with inhaled Isoflurane (0.5−3%), fentanyl (10−25 μg/kg), and pancuronium (1 mg/kg).
   NOTE: For this proof-of-principle study, juvenile Yorkshire pigs (14−42 days old, n = 18) were used that ranged from 2.5−10.5 kg body weight and 18−137 g heart weight (Figure 2). If an additional injection for induction is necessary, ketamine (10 mg/kg) can be injected I.M.
2. Once the animal is completely anesthetized and non-responsive, perform a sternotomy to expose the ascending aorta and right atrium.
   1. Using a scalpel, make a midline incision from the top of the sternum at the thoracic inlet, down to the xiphoid process. With a cautery (or scissors), dissect the underlying fat and muscle until the sternum is visible.
   2. From the xiphoid process, cut the sternum midline up through the manubrium with either surgical bone scissors or a bone saw. Insert retractors into the incision to expose the heart.
3. Deliver a bolus dose of heparin (300 U/kg) to the right atria, using an 18 G needle and syringe, to minimize blot clots upon organ excision. Place absorbent pads in the chest cavity and ice around the heart.
4. With scissors, carefully slice through the pericardium, isolate the aorta by blunt dissection from the surrounding connective tissue, and clamp the aorta just below the first arterial branch on the aortic arch. Using a 50 mL syringe with an 18 G needle, inject ice-cold cardioplegia (20 mL/kg) through the top of the ascending aorta.
5. Cut through the vessels leading to the heart and remove the heart with the ascending aorta intact and plunge the excised heart into ice-cold cardioplegia.
6. Grasp the walls of the aorta with a pair of hemostats and slip it onto a ribbed cannula attached to tubing leading to 1 L of ice cold cardioplegia media suspended above the heart (~95 cm to provide ~70 mm Hg). Allow fluid to enter and fill the aorta until it overflows to prevent any bubble from entering the vasculature.
   NOTE: Use of a mechanical uncoupler (2,3-butanedione monoxime [BDM] or blebbistatin) will decrease the coronary perfusion rate as the oxygen demand of the tissue declines.
7. Secure the aorta to the cannula using umbilical tape and further anchor it by tying up the hemostats to bear the weight of the heart, which is now hanging from the cannula (Figure 1C). Allow the cold media to retrograde perfuse the heart at a constant pressure of 70 mmHg via gravity. Keep the heart submerged in cold cardioplegia until ready to be transferred to the warmed (37 °C) Langendorff-perfusion system (<10 min).
   NOTE: The aorta on smaller hearts (<50 g, up to 2 week-old pig) will bear the weight of the heart but larger hearts are at risk of slipping off the cannula. During the initial cannulation and when moving to the warmed system, prevent air from entering the aorta, which can cause coronary emboli. Use large bore tubing (>3/8” internal diameter) which allows bubbles to rise faster than the solution entering the aorta.
8. Transfer the heart to the Langendorff system (37 °C) without introducing air into the cannula. Allow the normal sinus rhythm to flush the vasculature of any remaining blood and cardioplegia.
   NOTE: In the described study an average initial flow rate of 184 ± 17 mL/min was observed in isolated juvenile piglet hearts. The flow rate declined to 70 ± 7.5 mL/min (mean ± SEM) after perfusing with warmed media containing a mechanical uncoupler (20 mM BDM). Do not submerge the heart tissue, as it can impinge on cardiac imaging. Tissue temperature is maintained by coronary flow in the pig heart due to its larger volume and smaller surface area, as compared to rodents. Under full flow, the epicardium and endocardium temperatures ranged from 35 °C to 37 °C, respectively.
   CAUTION: Wear appropriate personal protective equipment, including eye wear when working with mechanical uncouplers. The heart can eject media rapidly and unexpectedly.
9. Defibrillate the heart in the event of shockable arrhythmias (ventricular tachycardia, ventricular fibrillation) by placing external paddles at the apex and base of the heart and delivering a single shock at 5 J, increasing in 5 J increments (or as selectable by the defibrillator) until 50 J, cardioversion, or un-shockable rhythm. Repeat shocks at 50 J as necessary.
   NOTE: In the presented study, 89% of preparations required defibrillation. After equilibration (~10 min), an average heart rate of 70 ± 4.5 bpm (mean ± SEM) was observed for juvenile piglet hearts (Figure 2).
10. Flush the heart with at least 1 L of modified Krebs-Henseleit media, without recirculating, to remove any residual blood and cardioplegia. Once the media runs clear through the heart, close the circulating loop to recirculate perfusate.

3. Electrophysiology Study

1. To record a standard lead II electrocardiogram (ECG) throughout the course of study, attach a 29 G needle electrode to the ventricular epicardium near the apex, with another electrode in the right atrium. Connect the positive and negative inputs of a differential bioamplifier to the apex and right atrium, respectively.
2. Attach one bipolar stimulus electrode on the right atria, and a second bipolar stimulus electrode to the lateral left ventricle for pacing purposes.
3. Pace the heart using an electrophysiology stimulator, with the initial current set to twice the diastolic threshold (1−2 mA) and a 1 ms pulse width^35,36.
   NOTE: If the stimulation fails to elicit a response, the pulse width may be increased up to 2 ms. More current (~10x) is needed with large coaxial electrodes (bipolar stimulation).
4. Identify the pacing threshold by applying a series of stimulus impulses (1−2 mA, 1 ms pulse width) at defined pacing cycle lengths (PCL) to ensure consistent stimulus response.
   NOTE: Once the intrinsic rate is established, the initial impulse train may begin at a slightly shorter PCL.
5. Perform extrastimulus pacing using either an S1−S1 or S1−S2 pacing train, in the latter a train of 6−8 impulses (S1) was followed by a single impulse (S2). Decrease the S2 PCL stepwise by 10 ms (i.e., 200 ms, 190 ms, 180 ms, etc.) until it fails to capture. Step up to the penultimate PCL (i.e., 190 ms) and decrease in 1 ms intervals to find the most precise PCL before the loss of capture (i.e., 184 ms).
   NOTE: The same stimulation parameters are used for both S1 and S2 (1−2 mA, 1 ms pulse width). See Figure 3 for representative examples, or previously published values on porcine heart electrophysiology measurements³⁷.
   1. To establish the ventricular effective refractory period (VERP), 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.
      NOTE: The refractory period is the shortest achievable S1−S2 coupling interval.
   2. To define the Wenckebach cycle length (WBCL), use the stimulus electrode on the right atrium to find the shortest S1−S1 interval at which 1:1 atrioventricular conduction propagates via the normal conduction pathway.
      NOTE: Failure to do so represents 2^nd degree heart block.
   3. To define the sinus node recovery time (SNRT), use the stimulus electrode on the right atrium to apply a pacing train (S1−S1) and measure the time delay between the last impulse in the pacing train, and the recovery of spontaneous sinoatrial node-mediated activity.
   4. To establish atrioventricular node effective refractory period (AVNERP), use the stimulus electrode on the right atrium to find the shortest S1−S2 coupling interval at which the premature atrial stimulation is followed by a His bundle potential that elicits a QRS complex, which signifies ventricular depolarization.

4. Optical Mapping of Transmembrane Voltage and Intracellular Calcium

NOTE: A mechanical uncoupler should be used to minimize motion artifacts during optical mapping and to avoid hypoxia^3,38,39,40. (-/-)Blebbistatin (5 µM circulating concentration) may be added slowly as a bolus dose of 0.5 mM in 5 mL of perfusate (100x of final concentration)⁴¹. Alternatively, BDM may be initially included in the perfusate media at a circulating concentration of 20 mM.

1. Prepare the voltage dye by dissolving 5 mg of RH237 into 4 mL of anhydrous DMSO. Dilute the dye aliquot with up to 5 mL of media and vortex. Slowly add RH237 (62.1 µg per 500 mL of perfusate) proximal to the aortic cannula.
   NOTE: The myocardial tissue may be re-stained with RH237, if needed, throughout the duration of the experiment.
2. Prepare the calcium dye by dissolving 1 mg of Rhod2-AM into 1 mL of anhydrous DMSO. Mix the dye with 50 µL of pluronic acid, place in a 37 °C sonicating bath for up to 10 min, and then dilute with up to 5 mL of media. Slowly add the calcium dye (50 μg per 500 mL of perfusate) proximal to the aortic cannula.
   NOTE: To ensure uniform dye staining, dyes should be added slowly (>30 s). Rhod-2AM takes up to 10 min to reach peak fluorescence, while RH237 stains the heart within a 1−2 min. Using the described dye loading, signal to noise ratio (SNR) ranges of ~42−86 and ~35−69 for voltage and calcium, respectively, can be expected. SNR values can be calculated as SNR = (Peak-to-Peak counts)/(Standard deviation during diastolic interval)⁴².
3. Position the imaging hardware (camera, image splitter, lens) as shown in Figure 1, to focus on an appropriate field of view.
   NOTE: The splitter is configured with a dichroic mirror (660+ nm) that passes RH237 and reflects Rhod2 emission spectra. High-transmission emission filters are used for the RH237 (710 nm long pass) and Rhod2 (585 ± 40 nm) emitted light (long pass ET710, see Table of Materials). A wide-pupil 50 mm/F0.95l lens is attached to the front of the image splitter. This configuration results in adequate emission light separation, as previously validated^43,44.
4. Connect the camera to a workstation and acquire images using selected software, with an exposure time of 0.5−2 ms. Perform image alignment with the aid of software that can split the desired regions, overlay, and display a gray-scale subtraction or pseudo-color addition to highlight misalignment (see Table of Materials for software option).
5. Turn off the room light to minimize fluorescence interference from ambient lighting. Test the LED lights (525 nm, 1.4 mW/mm²) prior to the start of imaging to ensure uniform and maximal epicardial illumination, as determined by the sensor well depth.
   NOTE: Each light is directed through an excitation filter (535 ± 25 nm). LED lights can be triggered manually before filming to maximize signal linearity. Emitted fluorescence from the epicardium is passed through the image splitter and emission filters. Split images are projected onto a high-speed sensor. The field of view of is approximately 12 cm x 10 cm, or 5.9 cm x 4.7 cm for each split image, depending on lens choice and distance from the heart.
6. For optical mapping studies, image the myocardium during sinus rhythm, ventricular fibrillation (Figure 4) or dynamic pacing (S1−S1, 1−2 mA, 1 ms pulse width) via a stimulation electrode positioned on the left ventricle (Figure 5). Begin with a pacing cycle length of 350 ms, and decrement by 10−50 ms to generate restitution curves (Figure 5E)^35,36.

5. Cleanup

1. Remove the heart from the system and drain all perfusate. Rinse the system tubing and chambers with purified water.
2. For routine maintenance, periodically rinse the system with detergent solution or a diluted hydrogen peroxide solution, as needed.

6. Data Processing

1. Confirm optical signal quality throughout the study by opening a video file, selecting a region of interest, and plotting mean fluorescence over time using an appropriate software package or custom algorithm.
2. Analyze imaging data as previously described^{23,33,43,45,46}, to quantify action potential and calcium transient temporal parameters, including activation time, voltage-calcium coupling time (difference between Vm and Ca activation times), and repolarization duration measurements.
   1. Apply thresholding to isolate fluorescent epicardial pixels and discard noisy background data.
      NOTE: Thresholding will simplify and speed up analysis across large videos.
   2. Spatially filter optical signals over an epicardial surface area with kernel sizes ranging from 3 mm x 3 mm to 5 mm x 5 mm, as seen in Figure 4 and Figure 5.
      NOTE: The latter will improve the SNR without distorting action potential features, calcium transient morphology, or overall contour of the wavefronts^19,47. This may be unnecessary if using a sensor with large pixels or if binning during acquisition.
   3. Temporally filter signals with a digital lowpass filter (e.g., 5th-order Butterworth) with a cutoff frequency between 100 and 75 Hz to eliminate insignificant signal content⁴⁵.
      NOTE: See Figure 5C for an example of representative processed traces.
   4. Apply drift removal and subtraction, via Nth-order polynomial fitting, to minimize the effects of photobleaching, motion, or other significant sources of variation.
   5. After processing and normalization of optical data across an entire video, calculate action potential and calcium transient parameters of interest. Determine activation time, defined as the time of maximum derivative during depolarization, and peak fluorescence in order to calculate repolarization percentage times and periods (action potential duration [APD] and [Ca²⁺]i duration [CaD], see Figure 5).
   6. After temporal parameters are calculated, generate isochronal maps to depict aspects of a single action potential or calcium transient across the entire imaged epicardial surface using custom algorithms^{23,33,43,45,46}.
      NOTE: See Figure 5D for an example.