Improved Preservation of the Rat Heart with Celsior Solution Supplemented with Cariporide Plus...

American Journal of Transplantation 2005; 5: 1820–1826Blackwell Munksgaard

Copyright C© Blackwell Munksgaard 2005

doi: 10.1111/j.1600-6143.2005.00967.x

Improved Preservation of the Rat Heart with CelsiorSolution Supplemented with Cariporide Plus GlycerylTrinitrate

Ling Gaoa, Mark Hicksb,d

and Peter S. MacDonalda,c,∗

aVictor Chang Cardiac Research Institute, Heart & LungTransplant, Sydney, New South Wales, AustraliabSt. Vincents Hospital, Clinical Pharmacology andToxicology, Sydney, New South Wales, AustraliacSt. Vincents Hospital, Heart & Lung Transplant, Sydney,New South Wales, AustraliadUniversity of New South Wales, School of Physiology &Pharmacology, Sydney, New South Wales, Australia∗Corresponding author: Dr. Ling Gao,[email protected]

Our aim was to investigate whether the additionof glyceryl trinitrate (GTN), a source of nitric oxide,and/or cariporide, a Na/H exchange inhibitor, to a com-mercial preservation solution (Celsior) improved andextended cardiac preservation. After baseline indicesof cardiac function (aortic flow, coronary flow, heartrate, cardiac output) were measured in an isolatedworking rat heart model, hearts were arrested andstored at 2–3◦C for 6 or 10 h in Celsior solution alone,Celsior supplemented with either 0.1 mg/mL GTN or10 lM cariporide or both. After storage, functionalmeasurements were repeated and recovery of eachparameter was expressed as a percentage of its pre-storage baseline.

After 6 h storage, recovery of cardiac function was sig-nificantly better in hearts stored in GTN- or cariporide-supplemented Celsior solution compared with Celsiorsolution alone. The beneficial effect of GTN was sig-nificantly abrogated in hearts perfused with gliben-clamide prior to storage. Significant recovery of car-diac function after 10 h storage was only observed inhearts stored in Celsior solution supplemented withboth GTN and cariporide. Combined supplementationwith GTN and cariporide extends the safe period ofstorage of the rat heart and may be a useful approachto enhancing preservation of the donor heart.

Key words: Heart preservation, Celsior solution,cariporide, glycery trinitrate, ischemia-reperfusioninjury

Received 23 November 2004, revised 10 March 2005and accepted for publication 30 March 2005

Introduction

Although heart transplantation has been well establishedas a therapy for patients with end-stage heart disease,effective long-term heart preservation remains a prob-lem that has restricted viable donor graft preservation to6 h during clinical heart transplantation. It is now recog-nized that ischemia-reperfusion injury sustained by the car-diac allograft during transplantation adversely affects bothshort- and long-term outcome. More importantly, of all thefactors associated with early or late allograft failure,ischemia-reperfusion injury is one of the few that isamenable to therapeutic intervention. Such is the complex-ity of the molecular and cellular mechanisms that medi-ate ischemia-reperfusion injury, it is unlikely that any sin-gle treatment will provide maximal protection to the heartduring ischemia and reperfusion. Rather a combination oftherapeutic approaches is likely to be required.

Previous work in our laboratory has demonstrated the ben-eficial effects of cariporide, a Na+/H+ exchange inhibitor,on the preservation of cardiac function following 6 h hy-pothermic storage of the rat heart in the extracellular-basedstorage solution that is currently used for clinical hearttransplantation at our institution (1). Experimentally, Cel-sior solution has been shown to provide equivalent or su-perior cardiac preservation to most other preservation solu-tions currently used for clinical heart preservation, such asSt. Thomas’ Hospital solution and University of Wisconsinsolution (2,3).

Glyceryl trinitrate (GTN) has been widely used in the treat-ment of ischemic heart disease for more than 100 years.The anti-ischemic effect of GTN is believed to be basedon drug-induced decrease in cardiac pre-load and after-load, improvement of coronary collateral flow, dilation ofstenotic coronary arteries and inhibition of platelet aggre-gation (4). More recently, Glyceryl trinitrate (GTN) has beenreported to be an effective adjunct to hypothermic hyper-kalemic cardioplegia in experimental and clinical situations.Baxter et al. reported that the addition of GTN to Celsiorsolution improved heart preservation in an abdominal ratheart transplant model (5). The vasodilator action of GTNis thought to be mediated by its conversion to nitric oxide(NO) or a NO-containing metabolite and the consequentincrease in guanosine 3′5′ cyclic monophosphate (cyclic

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Improved Preservation of the Rat Heart

GMP) (6). However, a direct myocardial anti-ischemic ef-fect of GTN independent of cyclic GMP action has beendemonstrated in isolated rat hearts (7). The mechanism ofthis cyclic GMP-independent effect may be via the activa-tion of KATP by GTN-derived NO (8).

The aim of the present study was to investigate whetherthe addition of glyceryl trinitrate and/or cariporide, a Na+/H+

exchanger inhibitor, to a commercial preservation solu-tion (Celsior®) improved and extended cardiac preserva-tion. The potential additive cardio-protective effects of thecoadministration of GTN and cariporide was evaluated dur-ing prolonged hypothermic storage. Furthermore, the opti-mal timing of GTN administration and cariporide were alsoevaluated during the course of the experiment.

Materials and Methods

Male Wistar rats weighing 320–380 g were used in the present study. Allprocedures were approved by the Animal Ethics Committee of the Gar-van Institute of Medical Research (Sydney, Australia). All animals receivedhumane care in compliance with the guidelines set down by the NationalHealth and Medical Research Council (Australia) and the ‘Guide for the Careand Use of Laboratory Animals’ (National Institute of Health, Bethesda, MD).Krebs-Henseleit solution (37◦C) was used for perfusion of the isolated ratheart. Its composition was as follows: NaCl 118.0 mM; KCl 4.7 mM; MgSO4

1.2 mM; KH2PO4 1.2 mM; NaHCO3 25.0 mM; glucose 11.0 mM. The com-ponents were dissolved in MilliQ water and the resultant solution was bub-bled continuously with Carbogen (95%O2/5%CO2) at 37◦C for an hour priorto the experiment. The final pH of the Krebs solution was between 7.3 and7.4. The perfusate was filtered through an in line filter (5 lm) during thecourse of pre-storage perfusion and post-storage reperfusion. Celsior so-lution (Imtix Sangstat, France) (Table 1) was used for arresting the heartand also as the storage solution for the arrested heart. Cariporide (HOE642)(Aventis Pharma, Germany) (10 lM) was added to perfusate and/or car-dioplegia as well as storage solution immediately before the start of ex-periment. Glyceryl trinitrate (BDL, Australia) (0.1 mg/mL) was added to thecardioplegia and storage solution immediately before use.

Isolated working hearts were studied by a technique described in detail inprevious reports from our group (1). Briefly, rats were anaesthetized with anintraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Af-ter bolus injection of heparin 500 IU via the renal vein, the heart was rapidlyexcised and arrested by immersion in chilled (2–3◦C) perfusion buffer. The

Table 1: Composition of Celsior® solution

Component Concentration

Mannitol 60 mmolLactobionic acid 80 mmolGlutamic acid 20 mmolSodium hydroxide 100 mmolCalcium chloride, 2H2O 0.25 mmol Ca++Potassium chloride 15 mmol K+Magnesium chloride, 6H2O 13 mmol Mg++Histidine 30 mmolGlutathione 3 mmolOsmotality 320 mosmol/kgpH 7.3

aorta was cannulated and immediately perfused retrogradely on a Langen-dorff perfusion apparatus with Krebs buffer at a hydrostatic pressure of100 cm H2O. During this time, a small incision was made in the left atrialappendage into which another cannula was inserted and tied off. This non-working preparation was run for 15 min and then converted to a workingsystem by switching the supply of perfusate from the aorta to the left atrialcannula at a hydrostatic pressure of 20 cm H2O (pre-load). The workingheart ejected perfusate via the aortic valve into the aortic cannula. The hy-drostatic pressure in the aortic cannula was maintained at 100 cm H2O(after-load) throughout the working phase for all rat hearts.

Aortic pressure was monitored in a side arm of the aortic cannula with apressure transducer (Ohmeda, Pty Ltd., Singapore). Aortic flow was mea-sured by an in line flowmeter (Transonics Instruments Inc. Ithaca, NY). Aor-tic pressure and flow were recorded using MacLab/4e (ADInstruments PtyLtd, Sydney, Australia) and heart rate was calculated from the flow trace.Coronary flow was measured by timed collection of the effluent drainingfrom the apex of the heart.

Experimental protocol

Hearts remained in the working phase for 15 mins prior to storage. Mea-surements of heart rate (HR), aortic flow (AF), coronary flow (CF) and car-diac output (CO) were made at 10 min after conversion to working modeand used as pre-storage baseline. Any hearts having a baseline aortic flowless than 35 mL/min, or heart rate less than 200 beats/min, or coronaryflow less than 10 mL/min were excluded at this stage. After collection ofbaseline hemodynamic data, the heart was arrested by infusion of Celsiorsolution (at 2–3◦C) into the coronary circulation for 3 min from a reservoir60 cm above the heart. All hearts were stored on ice (2–3◦C) in 100 mLof the same solution for 6 or 10 h. Following that hearts were then re-mounted on the perfusion apparatus and reperfused in Langendorff modefor 15 min. Hearts were then switched to working mode and the afore-mentioned indices of cardiac function were recorded at 1, 5, 10, 15, 20 and30 min. Recovery of each parameter was expressed as a percentage of itspre-storage baseline.

Experimental groups

Figure 1 graphically illustrates the various treatment groups. Hearts storedfor 6 h before reperfusion were divided into 7 groups: (1) 6 h control: heartsstored in Celsior solution only, (2) 6 h CAP: hearts stored in Celsior so-lution supplemented with cariporide (10 lM), (3) 6 h GTN: hearts storedin Celsior solution supplemented with GTN (0.4 M), (4) 6 h GTN + CAP:hearts stored in Celsior supplemented with both GTN (0.4 M) and cari-poride (10 lM), (5) 6 h GTN/GTN: hearts were perfused with Krebs solutionsupplemented with GTN (0.4 lM) prior to storage in GTN-supplementedCelsior solution, (6) 6 h GL: hearts were perfused with Krebs solution sup-plemented with 0.1 lM glibenclamide prior to storage in Celsior solutiononly and (7) 6 h GL/GTN: hearts were perfused with Krebs solution supple-mented with 0.1 lM glibenclamide prior to storage in GTN-supplementedCelsior solution. Hearts stored for 10 h before reperfusion were divided into5 groups based on the treatments received: (8) 10 h GTN: hearts stored inCelsior supplemented with GTN (0.4 M) only, (9) 10 h CAP: hearts stored inCelsior supplemented with cariporide (10 lM) only, (10) 10 h GTN + CAP:hearts stored in Celsior supplemented with both GTN (0.4 M) and cariporide(10 lM), (11) 10 h CAP/CAP: hearts perfused with Krebs solution contain-ing cariporide (10 lM) then stored in Celsior supplemented with cariporide(10 lM), (12) 10 h CAP/CAP + GTN: hearts perfused with Krebs solutioncontaining cariporide (10 lM) then stored in Celsior supplemented with bothGTN (0.4 M) and cariporide (10 lM).

Statistical analyses

All results were expressed as the mean ± the standard error of the mean.Repeated measure analysis of variance (ANOVA) was performed for all

American Journal of Transplantation 2005; 5: 1820–1826 1821

Gao et al.

Figure 1: A schematic illustration

of the experimental protocol. Ar-rows indicate times at which mea-surements of cardiac function weremade. CP = cardioplegia, CAP =10 lM cariporide, GTN = 0.4 M glyc-eryl trinitrate (unless where speci-fied), GL = 0.1 lM glibenclamide.

Table 2: Pre-storage baseline of cardiac functions (mean ± SD)

AF CF CO HRGroups (mL/min) (mL/min) (mL/min) (bpm) n

6 h Control 48 ± 4.7 20 ± 2.2 68 ± 4.8 255 ± 30.7 86 h CAP 53 ± 9.5 18 ± 2.7 72 ± 10.7 245 ± 29.0 66 h GTN 51 ± 11.4 21 ± 1.9 72 ± 11.6 248 ± 43.4 96 h GTN + CAP 46 ± 4.1 21 ± 1.4 67 ± 4.3 245 ± 34.1 66 h GL 50 ± 6.4 19 ± 1.0 69 ± 6.9 256 ± 24.6 66 h GL/GTN 44 ± 4.6 22 ± 3.5 66 ± 5.3 248 ± 46.8 810 h GTN 42 ± 8.1 19 ± 1.0 61 ± 8.7 234 ± 38.4 510 h CAP 55 ± 15.4 17 ± 3.4 72 ± 18.5 243 ± 57.7 510 h GTN + CAP 51 ± 10.0 18 ±3.0 68 ± 11.0 273 ± 16.0 610 h CAP/CAP 44 ± 8.7 21 ± 2.6 63 ± 9.8 271 ± 44.3 410 h CAP/GTN + CAP 57 ± 9.1 18 ± 1.5 75 ± 10.4 253 ± 36.6 5p-value p > 0.05

functional measurements with Statview System. If the repeated ANOVArevealed a significant interaction, the statistical significance between anytwo groups was determined by Fisher’s protected least significant differ-ence test. Differences were considered significant when p-value was lessthan 0.05.

Results

The pre-storage baseline measures of cardiac function forcontrol and all treatment groups are shown in Table 2. Therewere no significant differences between groups.

After 6 h storage, hearts were significantly better pre-served with GTN-, cariporide-, or combined GTN andcariporide-supplemented Celsior solution compared withCelsior solution alone. As shown in Figure 2, recovery ofaortic flow after 20 min reperfusion was 42 ± 7% (6 hGTN), 66 ± 4% (6 h CAP) and 60 ± 5% (6 h GTN + CAP)compared with 15 ± 8% (6 h control) (p < 0.01). Similarly,

recovery of coronary flow was 70 ± 8% (6 h GTN), 82 ±3% (6 h CAP) and 85 ± 3% (6 h GTN + CAP) versus 31 ±12% (6 h control) (p < 0.01), recovery of cardiac outputwas 50 ± 7% (6 h GTN), 70 ± 3% (6 h CAP) and 68 ± 3%(6 h GTN + CAP) versus 22 ± 9% (6 h control) (p < 0.05),and recovery of heart rate was 94 ± 8% (6 h GTN), 115 ±6% (6 h CAP) and 95 ± 6% (6 h GTN + CAP) versus 48 ±12% (6 h control) (p < 0.01). The differences in recoveryof hemodynamic parameters between 6 h GTN and 6 hCAP groups were nonsignificant (p > 0.05) The 6 h GTN +CAP group was shown to have faster recoveries in mostof the hemodynamic parameters compared to 6 h GTNor 6 h CAP groups (Figure 2), however differences werenot significant either (p > 0.05). No further improvementsin recovery of cardiac functions were obtained by the ad-dition of GTN (0.4 lM) to the pre-storage perfusate (6 hGTN/GTN) in comparison to the 6 h GTN group (p > 0.05,data not shown). The improvement in aortic flow recoveryobserved in the 6 h GTN group was completely abolished

1822 American Journal of Transplantation 2005; 5: 1820–1826


Figure 2: Recovery of cardiac function after 6 h hypothermic storage: Mean values of recovery were obtained over 30 min after

switching to working mode during post-storage reperfusion. Data are expressed as a percentage of their pre-storage baseline and areshown for hearts stored in: (1) Celsior only (6 h Control, n = 8), (2) GTN supplemented Celsior (6 h GTN, n = 9), (3) cariporide supplementedCelsior (6 h CAP, n = 6), (4) combined cariporide and GTN supplemented Celsior (6 h GTN + CAP, n = 6), (5) GTN supplemented Celsiorfollowing pre-storage treatment by glibenclamide (6 h GL/GTN, n = 8), or (6) Celsior only following pre-storage treatment by glibenclamide(6 h GL, n = 6). Error bars indicate a standard error of the mean (SEM). ∗p < 0.05 versus control. #p < 0.05 versus GL/GTN & GL (repeatedmeasures ANOVA).

Figure 3: Recovery of cardiac func-

tion after 10 h hypothermic storage:

Mean values of recovery were ob-

tained over 30 min after switching to

working mode during post-storage

reperfusion. Data are expressed as apercentage of their pre-storage valueand are shown for hearts stored in Cel-sior supplemented with: (1) GTN only(10 h GTN, n = 5), (2) Cariporide only(10 h CAP, n = 5), or (3) A combina-tion of both (10 h GTN + CAP, n = 6),or hearts subjected to cariporide pre-treatment followed by storage in Cel-sior supplemented with: (4) Cariporideonly (10 h CAP/CAP, n = 4), or (5) A com-bination of both (10 h CAP/GTN + CAP,n = 5). Error bars indicate a standard er-ror of the mean (SEM). ∗p < 0.05 versus10 h CAP, 10 h GTN. #p < 0.05 versus10 h GTN + CAP & 10 h CAP/CAP (re-peated measures ANOVA).

by the addition of glibenclamide (0.1 lM) to the Krebs per-fusate prior to storage (6 h GL/GTN group, Figure 2, upperleft panel). In comparison, the improvement of the coro-nary flow recovery observed in the 6 h GTN group was onlypartly inhibited by the addition of glibenclamide (0.1 lM) tothe Krebs perfusate prior to storage (6 h GL/GTN group,Figure 2, upper right panel). The pre-treatment of rat heartwith 0.1 lM glibenclamide followed by 6 h hypothermicstorage in Celsior solution without GTN supplementation

(6 h GL) resulted in poor recovery of all functional parame-ters which were, however, not significantly different fromthat with GTN supplementation (6 h GL/GTN).

After 10 h storage, poor recovery of all hemodynamic pa-rameters was observed in hearts preserved with Celsiorsolution containing either GTN or cariporide alone (10 hGTN, 10 h CAP, Figure 3), with no significant differencesbetween the two groups. The addition of 10 lM cariporide


Gao et al.

to the Krebs perfusate prior to 10 h storage in cariporide-supplemented Celsior solution (10 h CAP/CAP) was asso-ciated with significantly better heart rate recovery afterreperfusion (Figure 3, lower right panel), but did not im-prove other hemodynamic parameters. In contrast, signif-icant improvements in all hemodynamic parameters wereobserved when both GTN and cariporide were added tostorage solution. After 20 min post-storage reperfusion,functional recovery of the hearts stored with combinedGTN and cariporide supplementation (10 h GTN + CAP)were 17 ± 10% (AF), 49 ± 12% (CF), 25 ± 10% (CO) and53 ± 15% (HR), respectively. Differences were statisticallysignificant compared with hearts stored with either GTN orcariporide alone (p < 0.05, Figure 3). The best recovery ofcardiac function after 10 h storage was observed in thosehearts perfused with cariporide-supplemented Krebs solu-tion followed by storage in Celsior solution supplementedwith both GTN and cariporide (10 h CAP/GTN + CAP, Fig-ure 3). In this group, recoveries of hemodynamic parame-ters were as follows: aortic flow 24 ± 10%, coronary flow65 ± 2%, cardiac output 34 ± 8% and heart rate 105 ±10%. When compared to the 10 h GTN + CAP group, how-ever, only the difference in heart rate recovery (105 ± 10%vs. 53 ± 15% at 20 min post-reperfusion) achieved statis-tical significance (Figure 3, lower right panel).

Discussion

The major findings from the present study are as follows:(1) the addition of either GTN or cariporide to Celsior so-lution significantly improved the preservation of rat heartsafter 6 h of profound hypothermic storage, but did not ex-tend effective heart preservation to 10 h, (2) the protectiveeffect of GTN was largely inhibited by pre-treatment of theheart with glibenclamide, (3) supplementation of Celsiorsolution with both GTN and cariporide resulted in viable re-covery of cardiac function after 10 h hypothermic storage,(4) pre-treatment of the isolated rat heart with cariporide-supplemented Krebs solution produced only a small incre-ment in cardiac preservation to that achieved by the directaddition of cariporide alone or cariporide plus GTN to Cel-sior preservation solution and (5) pre-treatment of the rathearts with 0.4 lM GTN did not improve recovery of cardiacfunction further beyond that by adding GTN to the storagesolution.

A range of changes to the vascular endothelial functionhave been shown to be responsible for reperfusion injuryand organ failure (9). In particular, the reduction in nitricoxide synthase activity observed during hypoxia (10) mayresult in the observed loss of mechanical function andcoronary vasodilatory response to endothelium-dependentagents (11). In the setting of organ transplantation andpreservation, improved organ function has been observedwhen exogenous nitric oxide donating agents have been in-cluded in a range of hypothermic storage solutions in heartand lung models (5,12–15).

Although a thorough study of the mechanism by whichnitric oxide or GTN exerts its protective effects in thedonor heart was outside the scope of the present study,there are two potential possibilities for its action in ourexperimental model. First, the GTN may act at the levelof the guanylate cyclase enzyme, facilitating cyclic guano-sine monophosphate production and helping to maintaina dilated vascular bed during hypothermic storage (16). Asecond role for nitric oxide is its ‘direct’ cardioprotectiveeffects mediated by the activation of mitochondrial ATP-sensitive potassium channels (17). Chronic dosing of ratswith GTN demonstrated protection against myocardial is-chemia in an isolated Langendorff model that was inhib-ited by glibenclamide, a specific inhibitor of mitochondrialATP-sensitive potassium channels (8). In another study, iso-lated rat heart was perfused with nonvasodilatatory dosesof GTN (10−7 M) and cardioprotection effects were ob-served regardless of the existence of vascular toleranceto GTN (18). In the present study, pre-treatment of heartswith 0.1 lM glibenclamide completely suppressed the pro-tective effect of GTN on aortic flow, and partially blockedGTN’s protective effect on coronary flow. The results werethe same with or without the presence of GTN as wereshown by the similar recoveries of cardiac function in 6 hGL and 6 h GL/GTN groups (Figure 2). This may reflectthe relative contribution of ATP-sensitive potassium chan-nel activation in the preservation of myocyte function (con-tributing to aortic flow) and endothelial cell function (con-tributing to coronary flow).

In addition to evaluating the cardioprotective effects ofGTN added to preservation solution, the effects of GTN 0.4lM added to pre-storage perfusate prior to 6 h hypother-mic storage was also investigated in the present study.However, no further improvement in rat heart preserva-tion was observed beyond that achieved by adding GTN tothe preservation solution (data not shown). A 15 min pre-storage perfusion of the rat heart with a high concentrationof GTN (0.4 M) directly impaired rat heart function induc-ing significant bradycardia and reduced cardiac output. Thiswas consistent with previous findings that GTN produceda dose-dependent negative chronotropic effect (19).

Another mechanism believed to play an important role inischemia-reperfusion injury is the activation of Na+/H+ ex-changer (NHE). During myocardial ischemia, normal Na+

extrusion via Na+/K+ ATPase is decreased due to impairedenergy status. At the same time, the Na+/H+ exchanger isactivated by increased intracellular H+ due to intracellularacidosis. This results in continuing Na+ influx and eventu-ally Na+ overload and cellular edema. This, in turn, inducesa reversal of the Na+/Ca++ exchanger, which removes in-tracellular Na+ in exchange for extracellular Ca++. The endresult of Na+/H+ exchanger activation during ischemia is adangerous accumulation of Ca++, which may result in lethalcell injury. Since alterations in ion homeostasis are criticallyinvolved in the myocardial damage induced by ischemiaand reperfusion, pharmacological blockade of one or more



ports of Na+ entry has proved a promising approach to car-dioprotection. Cariporide, a Na+/H+ exchanger inhibitor, isone such approach.

Previous studies have shown significant cardioprotectiveeffects by cariporide in patients undergoing coronary arterybypass graft surgery (20). A cardioprotective effect of cari-poride on the cardiac allograft during heart transplantationhas also been shown in experimental studies (21–24). Fur-thermore, the cardioprotective effects induced by Na+/H+

exchange inhibitor are not antagonized by glibenclamide(25). The optimal timing of cariporide administration, how-ever, has been controversial. While some investigators re-ported improved heart preservation when cariporide wasadded directly to the storage solution (26,27), other investi-gators have observed little or no benefit with this approach(1,22). Using an orthotopic canine transplant model, Kimet al. observed significantly improved preservation of car-diac function only when cariporide was administered priorto induction of ischemia and prior to reperfusion (22). Sim-ilarly, a previous study in our laboratory showed that maxi-mal protection of the isolated rat heart was achieved whencariporide was given before storage and again before per-fusion (1). Furthermore, the addition of cariporide to thestorage solution in addition to pre- and post-treatment didnot result in any further improvement of cardiac function ineither study (1). In the present study, the addition of cari-poride to the preservation solution resulted in highly sig-nificant improvement of cardiac function following 6 h hy-pothermic preservation. This observation is consistent withthe findings of Knerr and Liebermann (28) who reported sig-nificant activation of Na+/Ca++ exchanger in isolated chickcardiac myocytes during hypothermic ischemia storage. Itcontrasts, however, with our earlier study in which cari-poride was added to a different extracellular preservation(St. Vincent’s Hospital, SVH) solution (1). We believe thatthe most likely explanation for this difference is that cardiacpreservation after 6 h storage in SVH solution was inferiorto that achieved with Celsior solution. The severe cardiacinjury sustained by rat hearts stored for 6 h storage in SVHsolution probably blunted or obscured any beneficial effectof cariporide added to the storage solution. Similarly, in thepresent study, we believe that the severity of the ischemicinjury sustained by hearts stored for 10 h in Celsior solu-tion masked the beneficial effects of cariporide (and GTN)that were observed in hearts stored for 6 h. The addition ofcariporide to both the Krebs perfusate (before storage) andthe Celsior solution in the present study enhanced cardiacpreservation compared with its addition to Celsior alone,however the increment in cardioprotection was small.

Repeat administration of cariporide prior to reperfusionmay have further enhanced its cardioprotective action asthere is evidence for further activation of the NHE at theonset of reperfusion (29). We decided not to do so, how-ever, in view of the results of the Expedition Trial (30). Inthis clinical study, repeated bolus administration of cari-poride to patients undergoing coronary bypass surgery was

found to result in a lower incidence of postoperative non-fatal myocardial infarction but an unexpectedly higher rateof stroke (30). It remains uncertain whether the increasedstroke rate in the cariporide-treated patients was a chancefinding, however it has raised a serious question regard-ing the safety of repeated bolus intravenous administra-tion of cariporide in patients undergoing cardiac surgeryand cardiopulmonary bypass. By only administering cari-poride prior to storage (equivalent to administration to thebrain-dead donor) and adding to the storage solution in thepresent study, potential recipient exposure was minimized.

A major finding of the present study was that the combinedaddition of cariporide and GTN to Celsior solution resultedin recovery of viable cardiac function after 10 h storage.This finding indicates an additive or synergistic interactionbetween GTN and cariporide. We and others have reporteda similar interaction between ischemic preconditioning andNa+/H+ exchange inhibition in the isolated rat heart (27,31).The importance of the present findings lies in the clinicalrelevance and applicability of the beneficial effects exertedby GTN, which is widely used in clinical practice in patientswith heart disease. Interestingly, the cardioprotective ef-fect of GTN was largely abolished by pre-treatment of theheart with glibenclamide, which has also been shown toblock the cardioprotective effects of acute ischemic pre-conditioning and other pharmacological agents that mimicthis phenomenon (32,33). The cardioprotection achievedby supplementation of Celsior solution with both GTN andcariporide, in contrast to the lack of protection achievedwith either agent alone, emphasizes the need to targetmultiple pathways in order to maximally protect the cardiacallograft from ischemia-reperfusion injury during prolongedhypothermic storage.

In conclusion, this study illustrated the enhanced myocar-dial preservation achieved by adding GTN or cariporide toCelsior solution during hypothermic heart storage. Resultsfrom the present study were consistent with previous ex-perimental studies indicating beneficial effects of eitherGTN or cariporide as single additives to storage solutionsfor heart preservation. In addition, this is the first studywhich has investigated the effects of combined GTN andcariporide supplementation during donor heart preserva-tion and an additive cardioprotective effect has been ob-served when both drugs were present. This suggests thatcoadministration of GTN and cariporide could be a usefulapproach to improve post-storage function and extend thesafe period of storage in donor hearts.

Acknowledgment

This study was supported by a grant from the National Heart Foundation ofAustralia (grant number G 04S 1619) given to Dr. Hicks.

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