Combining Cariporide with Glyceryl Trinitrate Optimizes Cardiac Preservation During Porcine Heart Transplantation

Authors


* Corresponding author: Peter Macdonald, pmacdonald@stvincents.com.au

Abstract

Sodium–hydrogen exchange inhibitors, such as cariporide, are potent cardioprotective agents, however, safety concerns have been raised about intravenously (i.v.) administered cariporide in humans. The aim of this study was to develop a preservation strategy that maintained cariporide's cardioprotective efficacy during heart transplantation while minimizing recipient exposure. We utilized a porcine model of orthotopic heart transplantation that incorporated donor brain death and 14 h static heart storage. Five groups were studied: control (CON), hearts stored in Celsior; CAR1, hearts stored in Celsior with donors and recipients receiving cariporide (2 mg/kg i.v.) prior to explantation and reperfusion, respectively; CAR2, hearts stored in Celsior supplemented with cariporide (10 μmol/L); GTN, hearts stored in Celsior supplemented with glyceryl trinitrate (GTN) (100 mg/L); and COMB, hearts stored in Celsior supplemented with cariporide (10 μmol/L) plus GTN (100 mg/L). A total of 5/5 CAR1 and 5/6 COMB recipients were weaned from cardiopulmonary bypass compared with 1/5 CON, 1/5 CAR2 and 0/5 GTN animals (p = 0.001). Hearts from the CAR1 and COMB groups demonstrated similar cardiac function and troponin release after transplantation. Supplementation of Celsior with cariporide plus GTN provided superior donor heart preservation to supplementation with either agent alone and equivalent preservation to that observed with systemic administration of cariporide to the donor and recipient.

Introduction

Ischemia-reperfusion injury to the donor heart is a major cause of primary allograft failure after heart transplantation. Current commercial preservation solutions incorporate a number of therapeutic approaches that are aimed at minimizing ischemia-reperfusion injury. The ability of these solutions to abrogate ischemia-reperfusion injury is limited, however, to the extent that donor heart ischemic times in excess of 4 h are associated with a progressive increase in the risk of primary graft failure and death after heart transplantation (1).

Sodium–hydrogen exchange inhibitors, such as cariporide, show considerable promise as cardioprotective agents in heart transplantation (2–7). The cardioprotective effects of cariporide appear to be greater when the drug is administered intravenously (i.v.) to the donor and recipient compared with adding the drug to preservation solutions (5,7), however, concerns have been raised about the safety of systemically administered cariporide following the report of an unexpectedly high fatal stroke rate in patients exposed to repeated high i.v. doses of cariporide while undergoing coronary bypass surgery (8).

We have reported that myocardial preservation in an isolated working rat heart model was markedly enhanced when both cariporide and glyceryl trinitrate (GTN), a nitric oxide donor, were added to Celsior (Genzyme Polyclonals, Lyon, France) preservation solution compared with either agent alone (9). Furthermore, the myocardial preservation achieved by combined supplementation was superior to that achieved by perfusion of the isolated heart with cariporide prior to storage and reperfusion. The aim of this study was to compare these myocardial preservation strategies in a porcine model of orthotopic heart transplantation. To further enhance the clinical relevance of our model, all donor animals were subjected to brain death and a period of post-brain death management that included combined hormonal resuscitation (10,11).

Materials and Methods

A porcine model of orthotopic heart transplantation incorporating donor brain death and hypothermic ischemic preservation was used (12–14). All procedures were approved by the Animal Ethics Committee of the Garvan Institute of Medical Research, Sydney, Australia (Animal Research Authority Reference No 04/26) and utilized in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (15).

Experimental groups

Twenty-six orthotopic heart transplants were performed in five treatment groups. Surgeons were not blinded to group allocation. In control experiments (CON; n = 5), donor hearts were flushed and stored in unmodified Celsior solution. Animals in the second group (CAR1; n = 5) were pretreated with cariporide (2 mg/kg i.v.) 15 min prior to donor cardiectomy and at 15 min prior to reperfusion in the recipient. The timing, dose and preparation of cariporide were chosen based on previous experiments (2). The third group of animals (CAR2; n = 5) had cariporide (10 μmol/L) added to Celsior solution and the fourth group (GTN; n = 5) had GTN (100 mg/L) added to Celsior solution. In the final group (COMB; n = 6), both cariporide (10 μmol/L) and GTN (100 mg/L) were added to Celsior solution. Aventis Pharma (Frankfurt am Main, Germany) provided cariporide for these experiments under an independent external investigator agreement.

Animals and anesthesia

Fifty-two juvenile Westran or Landrace pigs (weight range 40–60 kg) were obtained in pairs. Animals were premedicated with intramuscular ketamine (10 mg/kg), midazolam (1 mg/kg) and atropine (0.05 mg/kg). General anesthesia was induced using i.v. thiopentone (2–10 mg/kg). Animals were then intubated and ventilated with 100% oxygen. Anesthesia was maintained with isoflurane (1–4% inhaled gas) and fentanyl (100–150 μg i.v.) as required. Intravenous saline (0.9%) was infused at 10 mL/kg in the first hour, followed by 5 mL/kg/h titrated to central venous pressure (CVP) 0–5 mmHg. Continuous physiological monitoring of animals included: electrocardiogram, invasive arterial blood pressure (via a femoral arterial catheter), CVP (via an internal mammary central venous catheter), pulse oximetry, end-tidal expired carbon dioxide (CO2) and core temperature. Arterial blood gas and blood glucose analysis were performed at hourly intervals, and when clinically indicated using an i-STAT Portable Clinical Analyzer (i-STAT Corporation, East Windsor, NJ) and a Roche ‘Accuchek’ glucometer (Mannheim, Germany), respectively.

Donor surgery for brain death induction and data acquisition

A burrhole was made in the right frontoparietal skull and a Foley Catheter placed in the subdural space. Following administration of lignocaine (1 mg/kg i.v.) for arrhythmia prophylaxis, a median sternotomy was performed and the heart exposed. Arrhythmias were treated with additional lignocaine (1 mg/kg i.v.) and internal direct current defibrillation (10–30 J) if required. A flow probe (Transonic Systems Inc., Itaca, NY) was placed around the left anterior descending coronary artery (LAD) and a micromanometer-tipped catheter (Millar Instruments Inc., Houston, TX) was placed in the left ventricle. Six 2-mm diameter ultrasonic dimension transducers (Sonometrics Corp., Ontario, Canada) were sewn onto the epicardium to measure the base–apex major axis, anterior–posterior minor axis and the left ventricular free wall–right ventricular free wall minor axis diameters of the heart (12,14).

Brain death induction

After acquisition of baseline data, brain death was induced by inflating the Foley Catheter balloon with 24 mL water over approximately 3 min. This increased the intracranial pressure, causing an autonomic storm similar to that seen clinically. Anesthesia was ceased 15 min after balloon inflation. Brain death was confirmed by the typical hemodynamic changes of brain death, the absence of response to painful stimuli and the absence of brainstem (pupillary, corneal, gag and cough) reflexes following withdrawal of anesthesia (12).

Donor management

If despite i.v. fluid resuscitation, the mean arterial blood pressure (MAP) fell below 60 mmHg following brain death, an i.v. norepinephrine infusion (20 μg/mL) was commenced and titrated to achieve MAP of 60–70 mmHg. Three hours after brain death induction, a combined hormone resuscitation protocol (Table 1) (16) was commenced and continued for a further 3 h. Methylprednisolone was administered as a single i.v. bolus of 15 mg/kg. Tri-iodothyronine was administered as an initial i.v. bolus of 4 μg followed by a constant infusion of 4 μg/h. Vasopressin was commenced at 0.5 Units/h and increased incrementally to a maximum of 4 Units/h if required to maintain MAP of 60–70 mmHg. Attempts were made to wean animals off noradrenaline and if successful, attempts were then made to wean off vasopressin, while maintaining MAP 60–70 mmHg.

Table 1.  Hormone resuscitation protocol in brain dead donor animals
HormoneDose
Methylprednisolone15 mg/kg i.v. bolus
Triiodothyronine 4 μg i.v. bolus, followed by
Triiodothyronine 4 μg/h i.v. infusion
Vasopressin0.5–4.0 units/h titrated to mean arterial blood pressure 60–70 mmHg

Donor heart procurement

Six hours after brain death, a bolus intravenous injection of heparin (5000 IU) was administered. The superior vena cava was then ligated, an aortic cross-clamp applied and the heart arrested with 900 mL cold Celsior solution (Genzyme) infused into the aortic root from a 1-liter bag of Celsior solution (stored at 4°C prior to use) via a Medtronic (Sydney, Australia) DLP 12-gauge (9 Fr) aortic root cannula. The inferior vena cava and the left pulmonary vessels were transected to decompress the heart. The heart was then excised and stored in a sealed plastic bag containing 100 mL of Celsior solution in a container packed with ice. The total storage time (cold plus warm ischemia) was 14 h.

Orthotopic heart transplantation and recipient management

Recipient animals were prepared and anesthetized as described above. Methylprednisolone (500 mg i.v.) was given at anesthetic induction and at 15 min prior to cardiac reperfusion. A median sternotomy was performed and the left azygos vein was ligated extrapericardially. Heparin (10 000 IU) was given and the animal placed on cardiopulmonary bypass when the activated clotting time was greater than 500 s. Heparin 15 000 IU was added to the bypass pump prime and the animal was actively cooled to 32°C. Orthotopic transplantation of the donor heart was performed using the technique described by Lower and Shumway (17). Unmodified Celsior (200 mL) was infused into the coronary system via the aortic root after completion of the left atrial anastomosis and again after the right atrial anastomosis. Rewarming was commenced during the aortic anastomosis. Once the anastomoses were completed, the aortic cross-clamp was removed and the heart reperfused at 14 h ischemic time. The heart was deaired with a left ventricular (LV) vent and once warm, was defibrillated and paced (ventricular demand) at 120 beats/min. Dobutamine was commenced at 10 μg/kg/min, 45 min after reperfusion. This dose was chosen based on previous porcine transplant studies (2).

One hour after reperfusion, the first attempt was made to wean the animal from cardiopulmonary bypass. Successful weaning from bypass was defined as the transplanted heart being able to maintain MAP more than 50 mmHg for at least 20 min after weaning. If unsuccessful, the animal was placed back onto full bypass support and another attempt at weaning was made at 2 h postreperfusion. If still unsuccessful, dobutamine was increased to 20 μg/kg/min and a third attempt at weaning was made at 3 h. If still unsuccessful, the study was terminated. During all attempts at weaning CVP and LV end-diastolic pressure were monitored continuously to ensure adequate cardiac filling. Animals that were successfully weaned from bypass were monitored for a further 3 h with data acquired hourly (as described above). Blood loss during the study was autotransfused back into the animal via the bypass circuit.

Outcome measures

The primary outcome measures for the study were successful weaning from cardiopulmonary bypass and cardiac function postweaning from cardiopulmonary bypass. The left ventricular stroke work as determined by the area of the pressure volume loop was used to assess cardiac function before and after transplantation (14). LV stroke work data were recorded prior to brain death induction (baseline), and then at hourly intervals for 6 h prior to explantation of the donor heart. Data were also recorded 1 h after weaning from cardiopulmonary bypass posttransplantation and hourly thereafter for an additional 2 h. MAP, cardiac output (CO), LAD coronary blood flow and troponin I release posttransplant were also measured. Troponin I was assayed using the Bayer-Centaur Automated Chemiluminescence System (Bayer Healthcare Diagnostics, Tarrytown, NY).

Statistical analysis

Statistical analysis was performed using SPSS for Windows 13.0 (SPSS Inc., Chicago, IL). Continuous variables are reported as mean ± standard deviation and categorical variables as actual incidence/number of hearts in the study group. The number of hearts in each group that were successfully weaned from bypass was compared using Fisher's Exact test for a 2 × 5 contingency table. Study group baseline characteristics were compared using one-way analysis of variance (ANOVA). Changes in measured variables over time were compared between groups using two-way ANOVA. Significant differences in the ANOVA were investigated using Student's t-test for paired samples with a Bonferroni correction for multiple comparisons. Differences were considered statistically significant at p < 0.05.

Results

Selected baseline characteristics, total storage and warm ischemic times for the five experimental groups are shown in Table 2. There were no significant differences in any of the measured parameters between groups. The mean time between commencement of Celsior infusion and cardiac asystole at the time of donor heart procurement was 1.1 ± 0.4 min with no significant differences between treatment groups.

Table 2.  Characteristics of the control and treatment groups
CharacteristicCONCAR1CAR2GTNCOMBp-Value
  1. *ANOVA.

  2. Values are expressed as mean ± standard deviation.

  3. CON = control group; CAR1 = cariporide pretreatment group (n = 5); CAR2 = Celsior + cariporide group (n = 5); GTN = Celsior + GTN group (n = 5); COMB = Celsior + cariporide + GTN group (n = 6); LVV = left ventricular volume; BW = body weight.

Weight (kg)
 Donor42.9 ± 8.043.1 ± 7.652.4 ± 5.151.4 ± 8.750.3 ± 6.50.119
 Recipient40.5 ± 6.942.1 ± 4.448.2 ± 2.347.6 ± 7.247.0 ± 6.70.161
 Donor LV volume (mL)106 ± 13116 ± 17125 ± 19122 ± 21120 ± 130.423
 LVV/BW ratio 2.5 ± 0.2 2.7 ± 0.2 2.4 ± 0.4 2.4 ± 0.2 2.4 ± 0.40.550
Time (min)
 Brain death to explant380 ± 6 389 ± 19379 ± 3 375 ± 4 383 ± 6 0.210
 Warm ischemic time 52 ± 1048 ± 9 73 ± 22 58 ± 11 75 ± 270.077
 Total ischemic time846 ± 12836 ± 13856 ± 26861 ± 14851 ± 250.250

Weaning from cardiopulmonary bypass after heart transplantation

All 5 CAR1 and 5 of 6 COMB animals were successfully weaned from cardiopulmonary bypass compared with only 1 of 5 CON, 1 of 5 CAR2 and 0 of 5 GTN animals (p = 0.001). All transplanted hearts weaned from bypass were on 10 μg/kg/min dobutamine. CAR1 animals were weaned from cardiopulmonary bypass 122 ± 28 min (range 83–156 min) after commencing reperfusion compared with 120 ± 41 min (range 84–182 min) for COMB animals (p = ns).

Cardiac function pre- and posttransplant

Serial changes in the left ventricular stroke work (LVSW) over the course of the experiment are shown in Figure 1. There were no significant differences in baseline LVSW between groups. LVSW increased significantly in all groups at 3 h after brain death with no significant difference between groups (p < 0.001 vs. baseline). LVSW did not change significantly between 3 and 6 h after brain death. Following transplantation, the LVSW could not be assessed in the CON, CAR2 and GTN groups due to inability to wean these animals from cardiopulmonary bypass. In the CAR1 and COMB groups, the LVSW obtained postweaning was lower than the corresponding measurement obtained at 6 h post brain death, but not significantly different from the LVSW measured at baseline prior to brain death (Figure 1). LVSW remained stable over the 3 h postweaning and did not differ significantly between CAR1 and COMB groups at any time point.

Figure 1.

Calculated stroke work (SW) for the five treatment groups at baseline, 3 and 6 h after brain death (BD) and for the CAR1 and COMB groups over the first 3 h postweaning after heart transplantation: order of histograms from left to right is CON (n = 5), CAR1 (n = 5), CAR2 (n = 5), GTN (n = 5) and COMB (n = 6). SW increased significantly following brain death (p < 0.001 baseline vs. 3 h and 6 h post BD). SW could not be assessed in CON, CAR 2 and GTN groups posttransplantation due to failure to wean these animals from bypass. SW did not differ significantly between CAR1 and COMB groups at any time point.

Hemodynamic changes pre- and posttransplant

Systemic MAP data are shown in Figure 2. There were no differences in the MAP between groups during preexplantation and posttransplantation phases of the study. MAP was lower posttransplantation compared with baseline in both CAR1 and COMB groups, although the difference was significant only for the CAR1 group (50 ± 3, 50 ± 4, 48 ± 4 and 50 ± 5 mmHg at 0, 1, 2 and 3 h postweaning vs. 62 ± 5 mmHg at baseline for CAR1, p < 0.02; 56 ± 10, 54 ± 8, 56 ± 8 and 55 ± 6 mm Hg at 0, 1, 2 and 3 h postweaning vs. 62 ± 4 mmHg at baseline for COMB, p > 0.05). MAP did not change significantly during the 3 h after weaning off bypass in either group.

Figure 2.

Mean arterial blood pressure (MAP) for the five treatment groups at baseline, 3 and 6 h after brain death (BD) and for the CAR1 and COMB groups over the first 3 h postweaning after heart transplantation: order of histograms as for Figure 1. Postweaning data taken after successful weaning from cardiopulmonary bypass post heart transplant in CAR1 and COMB groups. Values are mean ± standard deviation for each time point. There were no significant differences between groups at any time point pre- or posttransplant. Posttransplant MAP was significantly lower than pretransplant MAP for CAR1 animals.

CO data are shown in Figure 3. There were no significant differences in CO between groups during preexplantation and posttransplantation periods. Posttransplant CO in both CAR1 and COMB groups tended to be higher compared with baseline but the differences were not statistically significant. CO did not change significantly in either group over the 3-h observation period after weaning from cardiopulmonary bypass.

Figure 3.

Cardiac output (CO) for the five treatment groups at baseline, 3 and 6 h after brain death (BD) and for the CAR1 and COMB groups over the first 3 h postweaning after heart transplantation: order of histograms as for Figure 1. Postweaning data taken after successful weaning from cardiopulmonary bypass postheart transplant in CAR1 and COMB groups. Values are mean ± standard deviation for each time point. There were no significant differences in CO between groups during preexplantation and posttransplantation periods. Posttransplant CO in both CAR1 and COMB groups tended to be higher than at baseline, however, the differences were not statistically significant.

LAD coronary artery flow pre- and posttransplant

LAD flow data are shown in Figure 4. Post hoc testing revealed a significant difference in LAD flow between GTN and COMB groups at baseline, but not between other groups. Over the 6 h of donor management, a trend to higher LAD flows was observed in the GTN group. This difference was significant at 6 h but not at 3 h post induction of brain death. Measurements of LAD flow postweaning from cardiopulmonary bypass after heart transplantation were only obtainable in the CAR1 and COMB groups. Measures of LAD flow posttransplant were significantly higher than pretransplant at all time points in both CAR1 and COMB groups (p < 0.0001), but there were no significant differences in posttransplant LAD flow between groups.

Figure 4.

Left anterior descending coronary artery (LAD) Flow for the five treatment groups at baseline, 3 and 6 h after brain death (BD) and for the CAR1 and COMB groups over the first 3 h postweaning after heart transplantation: order of histograms as for Figure 1. There was significant heterogeneity between groups at baseline and after induction of brain death with the GTN group having higher LAD flows at baseline and 6 h. LAD flow posttransplant was significantly higher than pretransplant in both CAR1 and COMB groups (p < 0.0001), however, there were no significant differences between the two groups.

Troponin I pre- and posttransplant

Troponin I data are shown in Figure 5. There were no significant differences between groups in plasma troponin I levels in the donor animals. Troponin I levels rose markedly in all groups over the first 3 h posttransplant (p < 0.0001). Differences between groups began to emerge as early as 15 m posttransplant, with the highest values recorded in the CON group and the lowest values observed in the COMB group (Figure 5). Troponin I was significantly higher in CON animals compared with COMB at 1-h postreperfusion (190.0 ± 46.1 vs. 80.7 ± 43.0 μg/L; p < 0.02) and at 3 h postreperfusion (895.6 ± 533.8 vs. 234.9 ± 102.2; p < 0.04). Troponin I levels were also significantly higher in the CON group compared with the CAR2 and GTN groups (but not with the CAR1 group) at 3 h postreperfusion (Figure 5).

Figure 5.

Plasma troponin I levels for the five treatment groups at baseline, 3 and 6 h after brain death (BD) and over the first 3 h postweaning after heart transplantation: order of histograms as for Figure 1. Plasma samples for troponin I could be obtained from all groups pre- and posttransplant. Transplanted hearts in the CON, CAR2 and GTN groups that could not be weaned from cardiopulmonary bypass were maintained on bypass to enable blood sampling to be taken until completion of the study protocol. Troponin I levels rose markedly in all groups over the first 3 h posttransplant (p < 0.0001). Significant differences between groups began to emerge as early as 15 min posttransplant with the highest values recorded in the CON group and the lowest values observed in the COMB group.

Discussion

The primary finding of this study was that supplementation of Celsior solution with both cariporide and GTN (COMB) resulted in preservation of the porcine heart during prolonged hypothermic storage and functional recovery posttransplantation that was equivalent to that observed when both donors and recipients had received i.v. cariporide (CAR1 group). In contrast, donor porcine hearts stored for the same period in Celsior solution alone or in Celsior solution supplemented with either cariporide or GTN showed poor recovery. Most of these hearts could not be weaned from cardiopulmonary bypass after orthotopic transplantation. Furthermore, troponin I release during the first 3 h posttransplant, which we have shown to be a sensitive marker of myocardial injury in this model (13), was lowest in the COMB group (Figure 5).

The changes in LV stroke work after transplantation in the COMB and CAR1 groups are complex and most likely reflect the opposing effects of ischemia-reperfusion injury (sustained during storage, implantation and reperfusion) and dobutamine (10 μg/kg/min), which was administered to all animals after transplantation. In the absence of ischemic injury, dobutamine at 10 μg/kg/min has been shown previously to increase contractility in the porcine heart by 120% (18). In this study, LVSW after transplantation in the presence of dobutamine was similar to the baseline LVSW obtained prior to brain death and exposure to dobutamine, indicating a markedly blunted response to dobutamine after transplantation relative to normal hearts (18). Nonetheless, LV contractile function in COMB and CAR1 animals was sufficient to maintain a viable circulation in these animals at all time points up to 3 h posttransplant.

These findings obtained in a porcine orthotopic heart transplant model match closely our findings reported previously in the isolated working rat heart (9). In that study, supplementation of Celsior solution with cariporide plus GTN provided superior cardiac preservation and recovery when compared with supplementation with either agent alone after hypothermic storage for up to 10 h in an isolated working rat heart model (9), consistent with a synergistic interaction between cariporide and GTN. The mechanism of this synergy remains unknown, but one possible explanation is combined activation (phosphorylation) of the prosurvival kinase ERK1/2 that constitutes one limb of the reperfusion injury salvage kinase (RISK) pathway (19) We have found that ERK1/2 is weakly phosphorylated by cariporide and GTN alone but strongly activated by the combination (20). This activation of ERK1/2 is associated with reduced apoptosis as assessed by cleaved caspase-3 expression on immunohistochemistry and Western blotting (21). Furthermore, the myocardial preservation achieved by combined supplementation of Celsior solution was superior to that achieved by perfusion of the isolated rat heart with cariporide prior to storage and reperfusion (9). It is possible that pretreatment of the donor animal with i.v. cariporide prior to storage of the donor heart in Celsior solution supplemented with both cariporide and GTN may have resulted in further enhancement of cardiac preservation, however, we did not explore this in this study because little is known regarding the impact of i.v. cariporide on other organs used for transplantation.

The porcine model used in this study was designed to mimic clinical heart transplantation. Porcine myocardium is similar to human myocardium in that it is relatively intolerant of ischemic preservation (22–24). We used pigs whose body weight was close to that of adult humans. All donor animals were subjected to brain death and managed for 6 h with a protocol incorporating ‘combined hormone resuscitation’, which has been shown to improve heart function and donor hemodynamics in both animal models and human subjects (16,25–28), and which has been recommended for human donor management (11,29). Finally, all hearts were transplanted orthotopically, and the primary outcome measure was the ability of the transplanted heart to support the circulation of the recipient after weaning from cardiopulmonary bypass.

Many experimental studies have demonstrated a powerful cardioprotective effect of cariporide and other NHE inhibitors in the setting of hypothermic ischemia and heart transplantation (2–7), but the optimal mode of delivery and timing of administration remain uncertain. Some studies have reported that the cardioprotective effect of cariporide is greater when the drug is administered intravenously to the donor and recipient compared with when the drug is added to the donor preservation solution (5,7), however, concerns have been raised regarding the safety of systemically administered cariporide following the report of an unexpectedly high fatal stroke rate in patients exposed to repeated high i.v. doses of cariporide while undergoing coronary bypass surgery (8). This concern regarding the potential toxicity of systemically administered cariporide prompted us to re-evaluate the use of cariporide as a supplement to existing preservation solutions (CAR2 and COMB groups). Although plasma cariporide levels were not measured in the CAR2 and COMB groups, it is likely that exposure of the recipient animals in these groups to cariporide would have been negligible because there was no direct administration of cariporide to the recipient and any cariporide-containing preservation solution was flushed from the heart with unsupplemented Celsior solution during implantation.

Previous studies have reported that the cardioprotective capacity of cariporide when added to existing preservation solutions appears limited (5,7), and this was confirmed in this study in which only one of five animals in the CAR2 group could be weaned from cardiopulmonary bypass posttransplant. It is unlikely that the addition of a higher concentration of cariporide to Celsior solution would have enhanced its cardioprotective action because the concentration of cariporide added to Celsior solution in this study (10 μmol/L) was the same as that which we found produced maximal cardioprotection in an isolated working rat heart model (7). Several investigators have shown that the cardioprotective capacity of cariporide can be markedly enhanced when combined with ischemic preconditioning (6) or with pharmacological agents that may mimic this phenomenon, including nitric oxide donors (9), adenosine agonists (30) and mitochondrial KATP channel agonists such as diazoxide and BMS-180448 (6,31). In this study, we chose to combine cariporide with GTN for a number of reasons. GTN is already widely used in clinical medicine, it has been shown to be cardioprotective in previous heart transplant studies (32,33) and we have previously demonstrated a synergistic cardioprotective action of cariporide plus GTN in an isolated working rat heart model (9).

Despite the significant insults of brain death and 14 h hypothermic storage, the transplanted hearts from the CAR1 and COMB groups were able to generate sufficient cardiac output to sustain stable circulatory hemodynamics in the transplanted animal. MAP in the transplanted animals after weaning from bypass tended to be lower than the pretransplant pressure in the donor, but the differences were small. The small fall in blood pressure posttransplant may be explained in part by the administration of dobutamine and isoflurane, both of which have vasodilatory properties.

A striking finding in this study was the increase in LAD flows (in both CAR1 and COMB) after weaning from bypass posttransplantation when compared with preexplantation flows. We believe this is most likely explained by reactive coronary hyperemia after prolonged ischemia (34). The significance of this finding is uncertain, but Budrikis and colleagues (34) reported that the intensity and duration of early reactive hyperemia after ischemic storage was related to the extent of preservation injury in an isolated blood-perused porcine heart model.

There are several limitations to our study. We used healthy juvenile pigs as donors. The positive findings obtained in this model may not translate to all human donors particularly those who are older or who have preexisting comorbidities. The duration of donor heart storage was considerably longer than that to which human donor hearts are exposed. We chose this storage time based on our previous finding in a similar model of orthotopic porcine heart transplantation that the donor heart was capable of viable recovery after 14 h storage when the donor and recipient had been treated with i.v. cariporide (35). We have also demonstrated that i.v. cariporide improves cardiac preservation at conventional ischemic times in this model (2), and so we think it is likely that the benefits observed in this study are likely to apply to donor hearts subjected to conventional ischemic times. We observed transplanted animals for only 3 h after weaning from cardiopulmonary bypass. It is possible that transplanted hearts that were successfully weaned from cardiopulmonary bypass may have failed at a later time point, but we think this is unlikely because cardiac function and circulatory hemodynamics remained stable throughout the 3-h observation period. Finally, surgeons were not blinded to treatment allocation. We believe that this is unlikely to have influenced the outcome, however, as a standardized protocol of weaning from cardiopulmonary bypass was used for all animals.

In summary, we have demonstrated in a large animal model of heart transplantation that incorporates brain death and optimal donor management that combined supplementation of Celsior preservation solution with cariporide and GTN permits viable recovery of the transplanted heart after 14 h ischemic storage. In addition, we have shown that posttransplant LV function is best preserved and troponin ‘leak’ is lowest when donor hearts are stored in Celsior solution supplemented with both cariporide and GTN. This combined preservation solution provides optimal myocardial protection during prolonged hypothermic storage while avoiding any potential hazards that might result from systemic administration of cariporide to the recipient.

Acknowledgments

The authors acknowledge the invaluable support for this project provided by the Clinical Perfusion Service at St. Vincent's Hospital led by Dr. Frank Junius AOM with the help of Andrew Dinale, Claudio Soto and Jonathon Cropper. They also thank Andrew Jabbour and Jireh Tsun for their technical support.

Conflict of Interest Statement

The authors have no conflicts of interest to declare in relation to this manuscript.

Funding Sources

This project was funded by the National Health and Medical Research Council of Australia (NHMRC Project Grant 354408). Dr. Hing was supported by a NHMRC Postgraduate Medical Scholarship, and a Sir Roy McCaughey Surgical Research Fellowship provided by the Royal Australasian College of Surgeons. Dr. Watson was supported by a Francis & Phyllis Thornell Shore Memorial Scholarship provided by the Royal Australasian College of Surgeons.

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