Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

Summary

A reliable noninvasive approach for functional assessment of the donor heart during normothermic ex situ heart perfusion (NESP) is lacking. We herein describe a protocol for ex situ assessment of myocardial performance using the epicardial echocardiography and conductance catheter method.

Abstract

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the allocation of a growing number of donor hearts with extended criteria. These marginal grafts are associated with a high risk of primary graft failure and may benefit from ex situ perfusion before transplant. This technology allows for extended organ preservation using warm oxygenated blood perfusion with continuous metabolic monitoring. The only NESP device currently available for clinical practice perfuses the organ in an unloaded non-working state, which does not allow for functional assessment of the beating heart. We therefore developed an original platform of NESP in working mode conditions with adjustment of left ventricular preload and afterload. This protocol was applied in porcine hearts. Ex situ functional assessment of the heart was achieved with intracardiac conductance catheterization and surface echocardiography. Along with a description of the experimental protocol, we herein report the main results, as well as pearls and pitfalls associated with the acquisition of pressure-volume loops and myocardial power during NESP. Correlations between hemodynamic findings and ultrasound variables are of major interest, especially for further rehabilitation of donor hearts before transplantation. This protocol aims to improve the assessment of donor hearts to both increase the donor pool and reduce the incidence of primary graft failure.

Introduction

Heart transplantation is the gold standard treatment for advanced heart failure, but is limited by current organ shortage¹. A growing number of donor hearts with extended criteria (age >45 years, cardiovascular risk factors, prolonged low flow, acute left ventricular dysfunction secondary to catecholaminergic storm) are allocated with an increased risk of primary graft failure². Moreover, hearts donated after controlled circulatory death (DCD) may be presented with myocardial injury secondary to prolonged warm ischemia³. Therefore, there is a need for a better assessment of these donor hearts before transplantation, especially to evaluate their eligibility for heart transplantation^4,5.

Normothermic ex situ perfusion (NESP) preserves the beating heart using warm oxygenated blood. The only commercially available device for NESP preserves the heart in a non-working state (Langendorff mode). This approach was initially applied to expand the preservation of the graft beyond the critical 4 h period of cold ischemia⁶. Another major advantage of this technology is to provide continuous assessment of myocardial viability based on lactate concentration in the perfusate⁶. However, this biochemical assessment has never been correlated with post-transplant outcomes to date. Similarly, Langendorff mode for NESP does not allow for hemodynamic and functional evaluation of the heart prior to transplantation. Some authors have reported the potential benefit of intracardiac catheterization during NESP to predict myocardial recovery after transplantation⁷.

The present report aims to provide a reproducible methodology to evaluate donor heart performance during NESP. We modified the circuit to allow for working mode perfusion and, therefore, for the acquisition of noninvasive functional variables with epicardial echocardiography. Myocardial work index, a load-independent variable, was recorded using pressure-strain loops. We investigated the relationships between myocardial work and hemodynamic variables obtained from intracardiac conductance catheterization.

Protocol

The present protocol was approved by the local ethics committee on animal experiments and by the Institutional Committee on Animal Welfare (APAFIS#30483-2021031811339219 v1, Animals Ethics Committee of the University of Paris Saclay, France). Animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals developed by the National Institute of Health and with the Principles of Laboratory Animal Care developed by the National Society for Medical Research.

NOTE: Surgical procedures were performed under strict sterility using the same techniques used for a human being. Experimental procedures included large white piglets (45-60 kg) and were performed under general anesthesia.

1. Animal conditioning and anesthesia protocol

1. Allow the animals to acclimate for 7 days, with congeners and environmental enrichment, to ensure animal welfare.
2. Do not feed the animals 12 h before their inclusion in the experimental protocol.
3. Perform a premedication 30 min before the procedure with an intramuscular injection of an equimolar mixture of tiletamine and zolazepam (10 mg/kg) in the neck muscles.
4. Once the animal is sedated, insert a catheter into the ear vein, and induce general anesthesia with an intravenous bolus of propofol (2 mg/kg) combined with administration of atracurium (2 mg/kg).
5. Intubate the animal with a 7.5 mm orotracheal probe.
6. Monitor the animal with continuous EKG, expiratory CO₂, and oximetry.
7. Maintain general anesthesia with inhaled isoflurane (2%) mixed with 40% oxygen supplement.

2. In situ hemodynamic and echocardiographic assessment of the heart

NOTE: Hemodynamic assessment is performed with a Swan Ganz Catheter, while baseline functional assessment of the heart is performed by transthoracic echocardiography.

1. Insert percutaneously an 8 French (Fr) sheath into the brachiocephalic venous trunk using the Seldinger technique⁸.
2. After de-airing the catheter and setting the 0 pressure, insert the Swan Ganz catheter into the 8 Fr sheath until a pulmonary pressure profile is observed on the monitoring screen.
3. Obtain the pulmonary arterial occlusion pressure by pushing the Sawn-Ganz catheter in the pulmonary circulation while the balloon is inflated.
4. Assess the cardiac output using the thermodilution approach by infusion of 10 mL of cold (4 °C) saline solution in the proximal line of the Swan Ganz catheter. Repeat the measurement three times.
5. Assess the left ventricular ejection fraction (LVEF) using the biplane Simpson technique⁹.
6. Explore the aortic valve and aortic root to identify any structural disorder or aortic regurgitation above grade 2 that could compromise ex situ perfusion of the heart through the ascending aorta (Figure 1).

3. Description and priming of the normothermic ex situ perfusion (NESP) machine

NOTE: A modified NESP module is used to alternatively perform Langendorff perfusion and working mode perfusion. Briefly, connect the aortic line of the circuit to a compliance chamber via a Y-connector. Add a pediatric oxygenator and a cardiotomy reservoir (70-80 cm height above the aortic connector of the module) to provide a left ventricle afterload of approximately 70 mmHg during the working mode. Connect another cardiotomy reservoir (7-10 cm height above the aortic connector of the module) to the main inflow line using a Y-connector to provide a left atrium preload of approximately 10 mmHg during the working mode (Figure 2). Coronary flow is assessed with a flow sensor connected to the pulmonary cannula. A centrifugal pump, a membrane oxygenator, and a heater-cooler machine are connected to the circuit (Figure 2). For solution descriptions, refer to Table 1.

1. Prime the perfusion circuit with the priming solution (Table 1).
2. Set the pump output at 1500 mL/min.
3. Add the blood retrieved from the donor pig (1200-1500 mL) in the circuit.
4. Set the gas mixer to achieve an oxygen partial pressure >250 mmHg.
5. Connect maintenance solution and adrenaline solution (Table 1) to the circuit and set the initial output respectively at 5 mL/h and 0.1 mL/h.
6. Set the temperature of the perfusate at room temperature (RT) before placement of the heart in the perfusion module.
7. During working mode, connect a syringe of dobutamine with a concentration of 2.5 mg/mL (output between 0.04-0.12 mg/h).

4. Heart procurement and instrumentation for normothermic ex situ heart perfusion

1. Heart procurement
   1. Place the animal in the supine position and continue to maintain general anesthesia.
   2. Perform a median sternotomy and open the pericardium.
   3. Suspend the pericardium with four stay sutures.
   4. Place 4-0 polypropylene sutures on the right atrium and on the ascending aorta to secure cannulations with tourniquets.
   5. After heparin infusion (300 UI/kg) and careful dissection of the aortic root, insert a double-staged venous cannula in the right atrium for blood collection and a single-lumen cannula in the ascending aorta for cardioplegia infusion.
   6. Isolate the superior and the inferior vena cava with Silastic tourniquets.
   7. Connect the venous cannula to a blood collecting bag containing 10,000 IU of unfractionated heparin.
   8. Place the piglet body in the Trendelenburg position to improve blood drainage into the collecting bag.
   9. After blood collection is completed, cross-clamp the ascending aorta, infuse Del Nido cardioplegia in the aortic root (Table 1), and check that the ascending aorta is under pressure (no aortic regurgitation).
   10. Unload the right and left atrium by opening the inferior vena cava and right pulmonary vein, respectively, while the superior vena cava is clamped by a tourniquet.
   11. Once cardioplegia infusion is completed, ligate the left hemiazygos vein with two stiches of 4-0 polypropylene.
   12. Proceed to heart procurement, keeping 2 cm of the pulmonary trunk along with the left atrium posterior wall.
   13. Verify that there is no patent foramen ovale by inspecting the atrial septum and close it if necessary using 4-0 polypropylene sutures.
2. Instrumentation of the heart before NESP
   1. Place the heart in a 4 °C saline solution and separate the ascending aorta from the pulmonary trunk. Verify that the aortic valve and the coronary ostia are not injured.
   2. Insert four pledgeted stitches (4-0 polypropylene) 5 mm below the distal section of the ascending aorta and insert the infusion cannula into the aorta. Tight a hose clamp around the aorta to secure the cannula.
   3. Insert a drainage cannula into the pulmonary trunk and secure with a 3-0 polypropylene running suture.
   4. Close the inferior and superior vena cava with 5-0 polypropylene running sutures.
   5. Close the left atrium posterior wall with a 4-0 polypropylene running suture.
   6. Insert a left vent cannula through the posterior wall of the left atrium wall and snare a tourniquet around.
   7. Insert a preload cannula into the left atrial appendage and snare a tourniquet around.

5. Connection to the NESP machine and resuscitation of the heart

NOTE: Before instrumentation of the heart, ensure that the materials necessary for resuscitation are available next to the perfusion circuit, especially a defibrillator with internal probes and an external pacemaker with epicardial electrodes. Ensure that the pressure line is connected to the aortic line, and that output sensor is placed on the coronary flow line. The afterload line must be clamped, as well as the preload line of the working mode circuit.

1. Decrease the pump flow to 200 mL/min.
2. Connect the heart to the aortic connector after de-airing the connector. Ensure that the heart is appropriately connected to the perfusion module so that the inferior ventricular walls and left and right atrium are in front of the operator. Avoid twisting the ascending aorta to prevent aortic regurgitation.
3. Adjust the aortic pressure to 30 mmHg at RT.
4. During resuscitation, perform a smooth cardiac massage until a sinus rhythm is restored.
5. Slowly increase the pump flow within 15-25 min by steps of 50 mL/min to achieve an aortic pressure of 65 mmHg. At the same time, increase the perfusate temperature by steps of 2-4 °C to reach 37 °C.
6. Once the aortic pressure is at 65 mmHg, and perfusate temperature is at 37 °C, deliver an electric shock at 5 J if needed, and repeat until the sinus rhythm is restored.
7. Secure an epicardial electrode on the right ventricular posterior wall and connect to an external pacemaker. Pace the heart at 80 BPM to overdrive spontaneous rhythm.
8. Connect the pulmonary cannula to the coronary flow line.
9. Perform arterial and venous blood samples for gas and biochemical analyses of the perfusate. Record the initial lactate concentration and correct biochemical disorders to achieve the following objectives: glucose >1 g/L, K⁺ 3.5-5.5 mmol/L, Ca²⁺ 1.0-1.20 mmol/L, pH 7.35-7.45, Na⁺ 135-145 mmol/L, and HCO3^- 20-24 mmol/L.
10. Adjust pump flow to reach a mean aortic pressure of 65-75 mmHg and coronary flow of 650-850 mL/min.
11. Perform arteriovenous blood gas analysis every 15 min to ensure that myocardial extraction of lactate is effective. If venous lactate is higher than arterial lactate, then increase mean aortic pressure to 80 mmHg by decreasing maintenance solution, and check the lactate concentration 15 min after. If arteriovenous lactate clearance is still impaired, then increase coronary flow to >850 mL and check lactate concentration 15 min later.

6. Working mode procedure

NOTE: Efficient arteriovenous clearance of lactate is usually achieved within 30 min after initiation of Langendorff perfusion. Working mode can then be initiated by connecting the preload cannula to the preload reservoir (this line was previously clamped during Langendorff mode). Similarly, the afterload line is connected to the aortic line (Figure 2). Set the flow sensor on the afterload line to measure cardiac output.

1. Open the preload line and adjust the pump flow to ensure stable filling of the preload reservoir. During this period, the left atrium and the left ventricle are progressively filled with blood.
2. Open the aortic afterload line and clamp the main line of the circuit used for Langendorff perfusion. The afterload reservoir is progressively filled-up. Ensure the drainage of the reservoir by an overflow line which brings the perfusate back to the main reservoir of the circuit.
3. Initiate the infusion of dobutamine at 0.04 mg/min.
4. Perform arterial and venous blood gas sample analysis to ensure that the myocardial extraction of lactate is still effective.
5. Once the cardiac output is stable, perform invasive hemodynamic assessment along with epicardial ultrasound measurements.

7. Pressure-volume (PV) loop assessment with the conductance method

NOTE: All calibration steps must be performed in working mode.

1. PV catheter placement into the left ventricle
   1. Clean the 7 Fr pigtail conductance catheter with saline solution and connect it to the hardware interface.
   2. Gently push the catheter into the introducer 8 Fr sheath previously inserted through the left atrium roof to be aligned with the mitral valve.
   3. As soon as the catheter crosses the mitral valve, adjust the appropriate position, considering optimal pressure and volume signals. If there is too much noise, gently move the conductance catheter to improve the quality of the loops.
2. PV loop catheter calibration
   1. Pressure calibration
      1. Once the conductance catheter is appropriately located in the left ventricle, open the calibration interface on the software and calibrate the pressure value using acquisition software for conductance measurements.
      2. Start recording, select 0 mmHg pressure and 100 mmHg on the control interface, and record for 5 s each.
      3. Then, stop recording and open the pressure calibration interface. Match the corresponding signal to the pressure level.
      4. Once calibrated, verify that the signal matches the values obtained by invasive blood pressure monitoring.
   2. Volume calibration
      1. Conductance calibration
         1. Open the control interface on the software for conductance measurements.
         2. Start recording, one after the other, select the volumes suggested by the calibration interface.
         3. Let the interface record for 5 s each, then stop recording.
         4. Use the data-trace obtained and open the volume calibration interface.
         5. Match the corresponding trace to the pressure level.
      2. Parallel volume calibration
         1. Surrounding heart tissue conducts electricity and contributes to the overall volume signal. Remove this parallel volume for accurate volume measurement (post-processing calibration).
         2. To assess parallel volume in this setup (myocardial wall), inject 10 cc of hypertonic saline solution (4%) into the left atrium line once.
         3. Do not repeat the operation to avoid hypernatremia.
3. Field correction factor calibration
   1. Enter the stroke volume value obtained from the ultrasound measurements.
      NOTE: Factor alpha will be calculated considering the ratio of stroke volumes obtained either by ultrasound measurements or conductance catheterization.
4. PV data collection
   1. Stop epicardial pacing of the heart to avoid interference with the conductance signal. Record data in a steady state when the signal is stabilized (Figure 3)
   2. Select a series of 10 consecutive loops and open the analysis software. The software will automatically provide stroke work, pre-recruitable stroke work, maximum dP/dt, minimum dP/dt, and tau index.
   3. To obtain the end-systolic pressure-volume relationship and end-diastolic pressure-volume relationship, record the signal during preload occlusion. Gradually clamp the atrial perfusion line until preload reduction is effective (Figure 4). Then slowly release the clamp.

8. Epicardial echocardiography assessment of the heart in a working state

1. Acquisition of ultrasound loops
   1. Place three EKG epicardial electrodes connected to the echocardiogram machine.
   2. Apply a sterile drape around the heart and use a transesophagus probe.
   3. Apply the probe to the upper wall of the left atrium and manually rotate the transducer until a four-chamber view is obtained (Figure 5).
   4. Start the echocardiographic acquisition software for myocardial performance assessment using the X-plan mode.
   5. Then, run the ultrasound probe motor to obtain three and two-chamber views. Analysis of these views allows for measurement of left ventricle ejection fraction and global longitudinal strain⁹.
2. Assessment of myocardial work index (MWI)
   1. Proceed to the acquisition of four-, three-, and two-chamber views and record simultaneous arterial pressure (Figure 6).
   2. Assess the global longitudinal strain using these views and open MWI software. Use the invasive blood pressure detected by the external sensor on the perfusion circuit during loop acquisition.
   3. Manually notify the software of the exact opening and closing timing of the aortic and mitral valves.
      NOTE: MWI software will automatically provide global MWI, constructive work, wasted work, and effective work.

Representative Results

We herein described a NESP protocol in a monoventricular working state, using a modified heart perfusion module usually employed in clinical practice for Langendorff perfusion of the donor heart before transplantation. This piglet model of NESP using the present custom module was developed in 2019. The modifications of the circuit were minor, as most of the perfusion circuit was re-used for experiments. The cap of the module provided a flexible and waterproof membrane to protect the heart during transportation. It also allowed surface echocardiography while it remained in a sterile environment. The recommended priming volume with mixed blood and priming solution is about 1200-1500 mL in clinical practice. In the present protocol, the priming volume was higher (2000 mL) because longer tubing and additional reservoirs were necessary for working mode perfusion. Therefore, such considerations required animals over 50 kg for a blood collection of >1500 mL.

Placement of the porcine heart in the perfusion module was different compared to previously reported models of NESP in working mode^10,11. Indeed, most of them described hearts suspended by the aorta, above a blood collection chamber, in a vertical position. In this protocol, we used a commercially custom module and set the heart with the anterior side laid in the perfusion box in a slightly tilted position and the posterior side facing the operator. However, Hatami et al. suggested that the heart's position during NESP was an important factor for optimal myocardial perfusion¹² and would be better than the hanging position.

The present protocol used six animals to perform experimental Langendorff mode (LM) for 30 min, followed by working mode (WM) perfusion for 2 h. Mean aortic pressure (MAP) and cardiac output (CO) were continuously monitored and recorded every 30 min. Cardiac power output (CPO) was calculated as follows: CO x MAP/451. Assessment of lactate concentration in the perfusate was performed every 30 min to ensure that myocardial extraction of lactate (MEL) was effective as evidence for myocardial viability during NESP. Hemodynamic assessment was performed as soon as possible at T0, T60, and T120 during WM perfusion. Metabolic and hemodynamic measurements during NESP are summarized in Table 2.

Considering hemodynamic assessment by cardiac catheterization, optimal PV loops were achieved with a conductance catheter placed through the left atrial roof, then crossing the mitral valve, with the pigtail placed in the apex of the left ventricle. The position of the conductance catheter was checked using epicardial echocardiography (Figure 5). The quality of PV loop signal may change depending on catheter position and interference with external pacing (Figure 7).

Functional assessment during working mode perfusion
Echocardiographic assessment during WM perfusion was performed in the custom setup used in this study and provided left ventricular ejection fraction (LVEF) assessment, global longitudinal strain (GLS), and myocardial work index (MWI) with reproducibility over the experiments. All three left ventricular views were obtained at any time point in all experiments (Figure 6). Mean LVEF, GLS, and MWI were 40.8 (± 11)%, -8.00 (± 2)%, and 652 (± 158) mmHg%, respectively. Conductance catheter measurements were performed during WM perfusion. Mean SW, maximum dP/dt, min dP/dt, end-systolic pressure-volume relationship (ESPVR), tau, and pre recruitable stroke work (PRSW) were respectively 877 (± 246) mmHg·mL, 1463 (± 385) mmHg/s, -1152 (± 383) mmHg/s, 5.13 (± 3.16), 79.4 (± 23) ms, and 63.4 (± 17.5) mmHg·mL during WM perfusion. Hemodynamic parameters assessed either by conductance catheter or by surface echocardiography during WM perfusion are summarized in Table 3 and Table 4.

A significant decrease in MWI was observed during WM perfusion over time in all experiments (Figure 8A), as well as cardiac output (Figure 8B) and other parameters related to ESPVR (Figure 8C). Global MWI was correlated with cardiac output measured by conductance catheter (r = 0.85, p < 0.001) (Figure 9).

Figure 1: Parasternal transthoracic echocardiography view of the aortic valve. The aortic valve and the ascending aorta are checked to ensure that there is no ascending aortic aneurysm and no significant aortic regurgitation above grade 2. The functional left ventricular ejection fraction is also assessed. Please click here to view a larger version of this figure.

Figure 2: Modified organ care system circuit for monoventricular working mode. (A) A compliance chamber is set on the afterload line to reproduce the vascular elasticity. A Y-connector is set in the principal arterial line to fill a reservoir at the height of 10 cm above the heart graft to provide a preload for the left atrium at 13-15 mmHg. Another Y-connector is placed on the principal arterial line before the aortic connector. (B) One of the branches of the Y-connector is connected to a 3/8 inch tubbing, connecting a pediatric oxygenator and a reservoir at the height of 70 cm to provide an afterload of the left ventricle of 60 mmHg Please click here to view a larger version of this figure.

Figure 3: Stable conductance signal provided by the pressure-volume conductance catheter. A stable signal of the pressure-volume loops recorded in the software is provided by a central position of the catheter inserted in the left ventricle through an 8 Fr sheath set into the left atrium. Please click here to view a larger version of this figure.

Figure 4: Progressive cross-clamping of the preload reservoir. The procedure of progressive occlusion of the tubbing from the preload reservoir and the left atrium provides a decrease in the volume injected into the left atrium. The pressure-volume loops are then recorded with the acquisition software. Please click here to view a larger version of this figure.

Figure 5: Transesophagus echographic probe position during the surface echocardiographic assessment of the heart graft during WM. (A) The probe is placed on the left atrium wall while the posterior face of the heart is facing the operator during NESP. (B) Such placement provides an echocardiographic view of the left atrium, the left ventricle, and the mitral valve. Please click here to view a larger version of this figure.

Figure 6: Left ventricular views obtained with TEE probe during NESP. The epicardial echocardiography using a transesophagus probe set on the posterior wall of the left atrium provides a two-chamber view of the left atrium and the left ventricle. Please click here to view a larger version of this figure.

Figure 7: Examples of poor conductance signal acquisition. (A) No-central positioned conductance catheter with signal disturbed by the movements of the ventricular septum. (B) Conductance signal disturbed by external pacing. Please click here to view a larger version of this figure.

Figure 8: Linear regression over time during WM perfusion. (A) Myocardial work index (MWI, mmHg%), (B) cardiac output (CO, mL.min^-1), and (C) end-systolic pressure-volume relationship (ESPVR). Please click here to view a larger version of this figure.

Figure 9: Relationship between MWI and cardiac output during working mode perfusion. Correlation curve between the myocardial work index (mmHg %) and the cardiac output (mL·min^-1) during ex situ heart perfusion in working mode. Please click here to view a larger version of this figure.

Priming solution	Maintenance solution	Adrenaline solution	Del Nido cardioplegia
500 mL NaCl solution	60 mg of Adenosine	0.25 mg of adrenaline	500 mg of Ringer solution
150 mg of Magnesium	40 mL of NaCl solution	500 mL of Glucose 5%	10 mL of KCl 10%
250 mg of Methylprednisolone	(concentration: 1.5 mg/mL)		3 mL of Xylocaïne 2%
1 g of Cefotaxime			6 mL of Mannitol 20%
			6 mL of Sodium Bicarbonate 8.4%
			7 mL of Magnesium Sulfate 15%

Table 1: Solution descriptions. The table provides the volumes and concentrations of constituents used to prepare the priming, maintenance, adrenaline, and Del Nido cardioplegia solutions used in this protocol. The Del Nido cardioplegia solution is used to achieve cardiac arrest along with myocardial protection during cold ischemic time. The priming solution is infused in the perfusion machine, along with the blood collected during the experimental protocol. The maintenance solution and the adrenaline solution are infused during the ex situ heart perfusion to maintain stable perfusion parameters.

	T0	T120
Lactate concentration (mmol/L)	2.4 (0.97–2.83)	1.27 (0.36–2.48)
Myocardial Extraction of Lactate (mmol/L)	0.15 (0.14–0.19)	0.08 (0.04–0.09)
pH	7.37 ( 7.31–7.45)	7.41 (7.31–7.47)
Potassium (mmol/L)	4.6 ( 4.4–5.1)	4.9 (4.3–5.5)
Systolic aortic pressure (mmHg)	132.5 (101.0–142.3)	101.0 (96.2–109.3)
Mean aortic pressure (mmHg)	97.5 (73.0–106.8)	77.0 (69.0–85.5)
Coronary Flow (mL/min)	925 (550–1050)	700 (550–875)
Cardiac Power Output	326.5 (116.5–485.5)	228.0 (185.5–361.0)

Table 2: Hemodynamic and metabolic parameters assessed during WM perfusion. Data are provided with the median and interquartile range.

	SW (mmHg·mL)	maximum dP/dt (mmHg/s)	min dP/dt (mmHg/s)	ESPVR	Tau (ms)	PRSW
Mean	877	1463	-1152	5.13	79.4	63.4
Median	816	1423	-1025	4.01	73.9	62.8
Standard deviation	246	385	383	3.16	23.0	17.5
Minimum	528	778	-1856	2.19	52.0	40.0
Maximum	1244	2119	-755	13.8	134	101

Table 3: Mean and median values obtained by the conductance catheter method during WM perfusion. Abbreviations: ESPVR: end-systolic pressure-volume ratio; PRSW: pre-recruitable stroke work; SW: stroke work.

	GLS (%)	LVEF (SB)	MWI	GCW
Mean	-8.04	40.8	652	936
Median	-8.00	37	642	919
Standard deviation	2.03	11.0	158	208
Minimum	-11.5	27	389	579
Maximum	-5.00	59	898	1268

Table 4: Mean and median values obtained by surface echocardiography during WM perfusion. Abbreviations: GLS: global longitudinal strain; LVEF: left ventricular ejection fraction; MWI: myocardial work index; MWE: myocardial work efficiency; GCW: global constructive work.

Discussion

There are some critical steps to consider in the NESP protocol. In situ preliminary assessment of the heart remained important, especially considering the aortic valve that should not present with significant aortic regurgitation (grade 2 or more); otherwise, the resuscitation of the heart will be compromised during the Langendorff period because of impaired coronary perfusion and myocardial ischemia. The initiation of the WM after Langendorff perfusion was a challenging maneuver, requiring at least two persons to regulate the filling of the preload reservoir, the pump flow, the pressure of the left atrium, and the aortic outflow line. This transition period was performed once metabolic effective myocardial extraction for lactate was achieved. During this period, the perfusion circuit could stop because of pump defusing related to major air embolism. The optimal placement of the ultrasound probe on the left atrial wall to obtain steady two- and three-chamber views was partially disturbed by the cumbersome cannulas and materials set around the heart. Echocardiographic data had to be recorded with a very steady ultrasound signal, with at least three contraction cycles.

Resuscitation of the heart during NESP is not explicitly reported in the literature. Only a few studies describe in detail the resuscitation procedure to initiate NESP¹³. Preliminary resuscitation approaches were developed in this protocol to achieve an optimal technique for resuscitation, including progressive reperfusion, by slowly increasing coronary flow and blood temperature (from room temperature to 37 °C). The main issue for ultrasound imaging was finding the optimal location for the probe on the left atrial roof. The position of the perfused heart, with its posterior wall facing the operator, allowed performing surface echocardiography without moving the heart and without the risk of aortic valve regurgitation. The presence of bubbles in the circuit altered imaging quality, and this problem must be avoided as much as possible. Optimization of the circuit was performed to reduce blood turbulences, especially considering blood drainage from the afterload reservoir to the main reservoir. A non-steady position of the conductance catheter into the left ventricle provided poor quality PV loop curves. The PV loop signal could however be significantly improved by introducing the catheter in the center of the left atrial posterior wall, through the center of the mitral valve, and positioned in the middle part of the left ventricle.

Loading left heart cavities is essential for ex situ echocardiographic evaluation. Even if the decline of the cardiac output has been previously described in other studies while the lactate trend remained stable, only a few articles described such consideration using a real monoventricular working mode perfusion¹¹. Biventricular working mode perfusion was not performed in this model for technical reasons, because such a system is even more complex and cumbersome. However, the lack of working mode for the RV is questionable because of LV and RV interdependence, a confounding factor in LV assessment. The lack of right ventricular assessment may also be questionable since RV failure is a common complication after transplant, associated with high mortality. Potassium concentration constantly increased in the perfusate without the possibility of clearing it because no blood filtration membrane was included in our custom circuit. The main issue concerning this perfusion mode is the fact that the organ itself is isolated from the other organs that could regulate its metabolism and clear all the metabolites produced by the myocardial metabolism. Some authors have described a perfusion model that included a hemofiltration system to provide prolonged NESP in working mode¹⁴, with a significant decrease of myocardial edema at the end of perfusion, which certainly participates in the decline of the myocardial performances over time.

Myocardial hemodynamic and echocardiographic performances decreased in NESP during working mode in our experience, as well as cardiac hemodynamics recorded by conductance catheterization. This suggests that perfusion should not be considered as a preservative method for donor hearts before transplant. During WM, trends of biochemical were different compared to Langendorff mode. Myocardial extraction of lactate during WM was continuously effective, while hemodynamic performance decreased progressively. This finding suggests that the lactate trend may not be a relevant parameter for assessing the myocardial performance in WM, as previously observed in other studies¹⁵.

Functional assessment of the heart during NESP would be of great interest to clinicians. Invasive assessment methods (PV loop technique) present several limitations. Indeed, the conductance technique should be considered to carefully draw reliable results, because of the isolation of the heart graft without a physiological biological environment that usually conduces the electric signal along with the myocardium itself¹⁶. The decision to transplant marginal grafts preserved with NESP technology is currently based only on lactate trends¹⁷. We trust this approach could be easily applied to resolve this major issue before transplantation. It can provide both anatomical (valvular disease, myocardial thickness) and functional assessments of the donor heart. Echocardiographic assessment of the left ventricle was achieved in the preclinical model and allowed to obtain MWI, a load-independent parameter that was significantly correlated to cardiac output assessed by a conductance catheter. These preliminary results highlight the role of surface echocardiographic evaluation during NESP in a working mode.

Materials

Name	Company	Catalog Number	Comments
3T Heater Cooler System	Liva Nova, Châtillon, France	IM-00727 A	Extracorporeal Heater Cooler device
4-0 polypropylene suture	Peters, bobigny, France	20S15B	sutures
5-0 polypropylene suture	Peters, bobigny, France	20S10B	sutures
Adenosine	Efisciens BV, Rotterdam, Netherlands	9088309	Drugs for the ex-vivo perfusion
Adrenaline	Aguettant, Lyon, France	600040	Drugs for the ex-vivo perfusion
Atracurium	Pfizer Holding France, Paris, France	582547	Drugs for the induction of the anesthesia
DeltaStream	Fresenius Medical Care, L’Arbresle, France	MEH2C4024	Extracorporeal blood pump
EKG epicardial electrodes	Cardinal Health LLC, Waukegan, Illinois, USA	31050522	EKG detection electrodes
External pacemaker	Medtronic Inc. Minneapolis, Minneapolis, USA	5392	Pacemaker device
Glucose 5%	B.Braun Melsungen AG, Melsungen, Germany	3400891780017	Drugs for the priming solution
Heart Perfusion Set, Organ Care System	Transmedics, Andover, MA, USA	Ref#1200	Normothermic ex-vivo heart perfusion device
Intellivue MX550	Philips Healthcare, Suresnes, France	NA	Permanent monitoring system
Istat 1	Abbott, Chicago, Ill, USA	714336-03O	Blood Analyzer machine
Labchart	AD Instruments Ltd, Paris, France	LabChart v8.1.21	Pressure Volume loops aquisition software
Magnesium	Aguettant, Lyon, France	564 780-6	Drugs for the cardioplegia
Magnesium Sulfate	Aguettant, Lyon, France	600111	Drugs for the cardioplegia
Mannitol 20%	Macopharma, Mouvoux, France	3400891694567.00	Drugs for the cardioplegia
Methylprednisolone	Mylan S.A.S, Saint Priest, France	400005623	Drugs for the priming solution
Millar Conductance Catheter	AD Instruments Ltd, Paris, France	Ventri-Cath 507	Pressure Volume loops conductance catheter
MWI software	General Electric Healthcare, Chicago, Ill, USA	NA	software used for the Ultrasound echocardiographic machine
Orotracheal probe	Smiths medical ASD, Inc., Minneapolis, Minneapolis, USA	100/199/070	probe for the intubation during anesthesia
Potassium chloride 10%	B.Braun Melsungen AG, Melsungen, Germany	3400892691527.00	Drugs for the cardioplegia
Propofol	Zoetis France, Malakoff, France	8083511	Drugs for the induction of the anesthesia
Quadrox-I small Adult Oxygenator	Getinge, Göteborg, Sweden	BE-HMO 50000	Extracorporeal blood oxygenator
Ringer solution	B.Braun Melsungen AG, Melsungen, Germany	DKE2323	Drugs for the cardioplegia
Sodium Bicarbonate	Laboratoire Renaudin, itxassou, France	3701447	Drugs for the cardioplegia
Sodium chloride	Aguettant, Lyon, France	606726	Drugs for the priming solution
Swan Ganz Catheter	Merit Medical, south jordan, utah, USA	5041856	Right pressure and cardiac output probe
Tiletamine	Virbac France, Carros, France	3597132126021.00	Drugs for the induction of the anesthesia
Transesophagus probe (3–8 MHz 6VT)	General Electric Healthcare, Chicago, Ill, USA	NA	Ultrasound echocardiographic transesophagus probe
Vivid E95 ultraSound Machine	General Electric Healthcare, Chicago, Ill, USA	NA	Ultrasound echocardiographic machine
Xylocaïne 2%	Aspen, Reuil-malmaison, France	600550	Drugs for the cardioplegia
Zolazepam	Virbac France, Carros, France	3597132126021.00	Drugs for the induction of the anesthesia