Oxygen Consumption of the Aerobically-Perfused Cardioplegic Donor Heart at Different Temperatures

Background

The criterion standard for clinical preservation of donor hearts for transplantation consists of flushing the coronary vessels with a cold, crystalloid cardioplegic solution followed by static ischemic storage at 4°C [1]. Data from the International Society of Heart and Lung Transplantation (ISHLT) registry 948 heart transplantations between 2009 and based on 19 June 2014 reveal an increase in hazard ratio for 1-year mortality from 1.0 (i.e., no increased risk) after 3 hours and 20 minutes of cold ischemia to 1.2, 1.4, and 1.8 after 4, 5, and 6 hours, respectively [2].

A logical way of extending the safe preservation time of donor hearts during cold cardioplegic storage is to omit hypoxia by aerobic perfusion. Recently a study was published [3] demonstrating safe orthotopic transplantation of porcine donor hearts harvested 24 hours after total brain and brain stem death (decapitation between cervical vertebra 2 and 3). The donor hearts were perfused at 8°C with a cardioplegic nutrition solution with oxygenated erythrocytes for 24 hours and then orthotopic heart transplantation was done in ten recipient pigs; all pigs had excellent heart function during the 24-hour follow-up period. In another study [4] intact endothelial and contractile function of coronary artery was shown after 8 hours of preservation-perfusion with the same method. A clinical study is now ongoing with this preservation method and the first patient was successfully transplanted on the 31st of October 2017.

During heart surgery using extracorporeal circulation, after aortic clamping, the heart is deprived of coronary blood flow except for non-coronary collateral blood flow from mediastinal arteries connecting with the coronary arteries [5]. This collateral blood flow cannot be measured, and therefore makes it impossible to know the exact oxygen consumption of the hearts in situ. However, during perfusion of the explanted whole heart there is no non-coronary collateral blood flow, and by using the method described in the present study an exact calculation of the oxygen consumption of the whole heart can be made. By knowing the oxygen consumption of the whole heart at different temperatures the amount of perfusion needed can be estimated and unnecessary perfusion can be avoided, minimizing hemolysis and myocardial edema that may occur during long-term perfusion of cardioplegic hearts.

The aim of the present study was to measure the oxygen consumption of perfusion-preserved cardioplegic porcine hearts at 37, 30, 22, 15, and 8°C.

Material and Methods

ANESTHESIA AND ANIMAL PREPARATION:

Anesthesia was induced by means of an intramuscular injection of ketamine 20 mg/kg body weight (Ketaminol Vet. Intervet, Boxmeer, Netherlands), xylazine 100 mg (Rompun Vet, Bayer, Solna, Sweden), and atropine 0.5 mg (Atropine, Mylan AB, Stockholm, Sweden). Fentanyl 4 μg/kg body weight (Fentanyl B. Braun, Melsungen, Germany) and midazolam 0.4 mg/kg body weight (Midazolam Panpharma, Panpharma S.A., Trittau, Germany) were given intravenously through an ear vein before tracheostomy. Anesthesia and muscle relaxation were maintained by continuous intravenous infusion of a mixture of ketamine (10 mg/h/kg body weight) and rocuronium (1.5 mg/h/kg) (Fresenius Kabi, Uppsala, Sweden).

Volume-controlled and pressure-regulated ventilation was used, with a minute volume of 100–150 mL/kg body weight and a frequency of 20 breaths/minute. Positive end-expiratory pressure was adjusted to 5 cm H₂O and the inspired oxygen fraction was 0.5. Aortic pressure was measured with a catheter placed in the ascending aorta.

A median sternotomy was performed and the heart exposed. Heparin 500 IU/kg body weight (Heparin, LEO Pharma, Malmö, Sweden) was given intravenously. A cardioplegic pin was placed in the ascending aorta and after clamping the aorta, cardiac arrest was induced with St. Thomas cardioplegic solution (Plegisol, Pfizer, Sollentuna, Sweden). The heart was explanted and mounted in the heart perfusion system and perfusion was started within 15 minutes. The pulmonary artery and the superior and inferior caval veins were cut short and left open for coronary sinus blood to freely leave the heart. A silicon tube with side holes was placed through the left atrium into the left ventricle to drain the left heart in case aortic valve insufficiency should occur during perfusion.

THE PERFUSION SYSTEM:

The main components of the perfusion system are: 2 roller pumps, oxygenator, 2 heater-cooler units, a 3-liter zipper plastic bag and a water bath (Figure 1). One pump was used to perfuse the heart and the other pump was used to mix the perfusate. The plastic bag containing the heart was submerged in a temperature-controlled water bath to maintain a stable temperature. All tubes were wrapped in thermo-isolating material to decrease temperature exchange with the environment. Temperatures were measured in 3 places: 2 needle probes penetrated the perfusion tube and the mixing tube, respectively, and a regular probe was placed in the water bath. Perfusion pressure in the aorta was measured and recorded by a computer system. The venous blood from the whole heart was emptied into the reservoir, and to obtain a complete mixing, 500 mL/min was circulated in a separate circuit. Blood gas samples were taken from the mixed blood as indicated in Figure 1.

The perfusion solution has been described previously [3]. It is an albumin-containing hyperoncotic potassium-cardioplegic nutrition-hormone solution with washed and leucocyte-filtered erythrocytes from a compatible pig blood donor. The hemoglobin concentration of the perfusion solution was approximately 30 g/L in all groups, giving a hematocrit of approximately 10%. In earlier experiments we have found that immersed cardioplegic porcine hearts can be adequately perfused with a perfusion pressure of 20 mm Hg [3,4]. Twenty mm Hg in perfusion pressure gives a coronary flow of around 140 mL/min in hearts from pigs weighing about 50 kg.

The perfusion flow was set to 140 mL/min and kept at this level throughout the experiment in all groups. The mean perfusion pressure was 18 mm Hg (range 16 to 21 mm Hg). Initially the oxygenator was supplied with 95% oxygen and 5% carbon dioxide until the erythrocytes in the perfusate were fully saturated. The oxygenator was then excluded from the system. The perfusion was continued until the oxygen saturation of the erythrocytes in the perfusate fell to less than 20% (Figure 2). The pO2-values were then less than 2 kPa (Figure 3).

DESIGN OF THE STUDY:

The hearts were randomized into 5 groups, with each group exposed to one of the following temperatures: 37, 30, 22, 15, and 8°C. Blood samples were collected regularly for analysis in a blood gas machine (Radiometer ABL800 flex, Copenhagen, Denmark). Values for pH, PO2, and PCO2 were corrected for temperature by the machine. The perfusate volume and blood sampling intervals in the different groups are shown in Table 1. Due to the low oxygen consumption at 8°C, a smaller perfusate volume was used to limit the experimental time to about 3 hours. Similarly, due to the higher oxygen consumption at 37°C, a greater perfusate volume was used to avoid the experimental time being too short. On the days on which the experiments were conducted, the mean atmospheric pressure was 101.45±0.15 kPa, and the mean oxygen tension in the water-bath surrounding the plastic bag containing the heart was 21.30±0.10 kPa. Calculation of oxygen consumption was done during the period in which the reduction in oxygen saturation of the erythrocytes in the perfusate was linear, which was between 80% and 60% (Figure 2).

INVESTIGATION OF THE AIRTIGHTNESS OF THE PERFUSION SYSTEM:

As seen in Table 2, the PaO2 values in the SO2 interval between 80% and 60% were in the range of 7 to 1 kPa, whereas the ΔPO2 within each group was in the range of 1.7 to 0.63 kPa. The PO2 in the water-bath was around 21 kPa; thus, there was a possibility for leakage of oxygen from the water-bath into the perfusate if the plastic bag was not impermeable to oxygen. To investigate whether there was oxygen leakage into the perfusate during perfusion, separate experiments were performed. Using the same setup as described above, including erythrocyte-mixed perfusate but without a heart, a mixed gas consisting of 93% nitrogen and 7% carbon dioxide was supplied to the oxygenator to remove the oxygen from the perfusate. After oxygen saturation had decreased to less than 20%, the oxygenator was bypassed. Blood samples were taken every 10 minutes for blood gas analysis. A constant oxygen leakage (evidenced as re-saturation of the perfusate) was found for each temperature. When the oxygen saturation lay between 60% and 80% the oxygen leakage at 37, 30, 22, 15, and 8°C was 0.082, 0.072, 0.066, 0.060, and 0.042 mL/min, respectively. The results were adjusted for this leakage when calculating the oxygen consumption of the heart.

WEIGHT MEASUREMENT OF THE HEART:

The weight of the heart, including the weight of the ascending aorta and the aortic cannula, was measured before the heart was mounted in the system (W1). After the experiment had been completed, the aorta including the cannula and the pulmonary artery, were excised from the heart. The weight of the excised material was measured (W2). The heart weight (W) was calculated as: W1–W2.

DATA ANALYSIS:

The formula used to calculate oxygen consumption (MVO₂), given as mL/min/100 g heart weight, was:

where Δ is the difference between 2 consecutive samplings, SO2 is hemoglobin oxygen saturation, Hb is hemoglobin concentration (g/L), PO2 is the partial pressure of oxygen (kPa), W is the heart weight (g), volume is the perfusate volume (L), and time is the time in minutes between 2 consecutive samplings [6,7]. Results were expressed as the mean ± standard error of the mean, n=6 in each group.

Results

The weight of the hearts was 208±4 g (n=30).

The oxygen consumption given as mL/min/100 g heart tissue was at 37°C=1.10±0.04; 30°C=0.58±0.02; 22°C=0.33±0.01; 15°C=0.21±0.01, and 8°C=0.16±0.02 (Figure 4).

As seen in Figure 2, the rate of erythrocyte desaturation decreased with decreasing temperature. In the 8°C group it took 180 minutes to reach 20% saturation, even though the perfusate volume was 700 mL, whereas the 37°C group reached 20% saturation after only 40 minutes, even with a perfusate volume of 1200 mL.

Discussion

The domestic pig is the most commonly used large animal in cardiopulmonary research today. Domestic pigs are bred and managed to reach a body weight of 100–110 kg within 150–200 days of life. Even though the commercial life span of domestic pigs is only 6 months in meat production, their natural life span may be 15–25 years depending upon the breed and environmental circumstances. Due to the rapid growth of domestic pigs, their metabolic needs are greater than those of humans; the metabolizable energy intake in kcal/day for the indicated body weights is: 3300 (10–20 kg), 6100 (20–50 kg), and 8400 (50–80 kg) [8]. With this high energy intake and rapid growth, it is relevant to ask whether the oxygen consumption of domestic pig hearts is correspondingly high, since the clinical relevance of using pig hearts to study heart preservation is greater if such hearts have a metabolism similar to that of man. The pig heart anatomy differs slightly from that of the human heart. The left azygos vein in pigs drains its blood directly into the coronary sinus. To calculate the oxygen consumption based on the hemoglobin saturation in the coronary sinus, the left azygos vein must be ligated, otherwise a dilution of the coronary sinus blood with systemic vein blood will occur and make the calculation of the oxygen consumption uncertain.

Budrikis et al. [6] have studied the oxygen consumption of 60 kg domestic pigs at normothermia in a beating heart model with a mean cardiac output of 5.0±0.24 L/min, a mean coronary flow of 246±30 mL/min, and a mean left ventricular developed pressure of 145±9 mm Hg; they found the mean oxygen consumption of the heart to be 5.6±0.4 mL/min/100 g heart tissue. They calculated oxygen consumption based on the hemoglobin concentration and saturation in the ascending aorta and in the coronary sinus (with the left azygos vein ligated). Considering that the coronary sinus drains only around 85% of the whole heart, an estimated oxygen consumption in the range 6 to 7 mL/min/100 g seems reasonable based on adding 15% to the calculated value of 5.6 mL/min/100 g. In the present study, the oxygen consumption of the cardioplegic heart at 37°C was 1.1 mL/min/100 g, representing a reduction of 85% compared to a working normothermic porcine heart. At 8°C the oxygen consumption was only 0.16 mL/min/100 g, representing a 97% reduction compared to a working heart at normothermia [6].

In the study by Steen et al. [3] showing safe preservation of orthotopically transplanted porcine hearts after 24 hours of perfusion-preservation at 8°C, the hearts were perfused only 20% of the time in cycles of 15 minutes perfusion and 60 minutes non-perfusion; before start of each perfusion the SO2 in the coronary sinus had fallen from 97% to 23–30%, i.e., during the non-perfusion phase the heart received oxygen from the de-saturating red cells within the capillaries of the heart. With the low oxygen consumption at 8°C, the perfusion can be interrupted for 1 hour without myocardial hypoxia occurring. Besides minimizing the perfusion trauma, this makes the method safer during transport.

Interestingly, the oxygen consumption of the cardioplegic human heart at 37°C has been found to be within the range of 0.6 to 1.5 mL O2/min/100 g, at 22°C it is 0.31 mL/min/100 g, and at 10 to 12°C it is 0.135 mL/min/100 g [9]. This is close to what we found in the present study, indicating that the oxygen consumption of the cardioplegic pig heart does not differ significantly from that of the human heart. The pig heart is of special interest since it is the leading candidate as a donor heart in xenotransplantation [10].

Conclusions

The oxygen consumption of the perfused cardioplegic porcine heart at normothermia is 1.1 mL/min/100 g, at 22°C it is 0.33 mL/min/100 g, and at 8°C it is reduced by 85% to 0.16 mL/min/100 g.