SUMMARY

1. Ischaemic preconditioning (IP) can significantly reduce theextent of infarct size, contractile dysfunction and necrosis inhearts from a number of animal species. Activation of ATP-sensitive potassium channels has been implicated in this process.The aims of the present study were to determine the extent towhich IP preserves haemodynamic function in the rat isolatedworking heart model after prolonged hypothermic storage andto examine the involvement of activation of potassium channelsin this process.

2. Hearts from Wistar rats were perfused on a Langendorffapparatus. After stabilization in working mode, baseline meas-urements of heart rate, aortic flow, coronary flow and cardiacoutput were performed. Hearts were randomized to one of sixtreatment groups: (i) untreated control; (ii) IP; (iii) 3 min per-fusion with 200 mmol/L pinacidil; (iv) pinacidil vehicle; (v) 3 minperfusion with 10 mmol/L glibenclamide before IP; and (vi) 3 minperfusion with glibenclamide then pinacidil. Hearts were storedin an extracellular-based preservation solution for 6 or 12 h at2–3°C, remounted on the perfusion apparatus, stabilized asbefore and then haemodynamic measurements were repeated,after which time heart water contents were determined.

3. Recovery of haemodynamic function was markedly en-hanced in the IP and pinacidil-treated groups compared withuntreated and vehicle controls. These beneficial effects were com-pletely blocked by glibenclamide. These results suggest thatstrategies for activating potassium channels in donor hearts mayprotect organs during hypothermic storage prior to transplan-tation.

Key words: ATP-sensitive potassium channels, glibenclamide,heart transplantation, ischaemic preconditioning, myocardialreperfusion injury, organ preservation, pinacidil.

INTRODUCTION

Periods of organ ischaemia and reperfusion are mandatory duringthe procurement, storage and implantation phases of the transplan-tation process. The major challenge to preservation of donor organsis to minimize any accompanying pathological damage and impair-ment of function. Current clinical practice used in heart preservationvaries widely, but is based on single flush induction of cardioplegiaand hypothermic storage in one of a number of storage solutions.1

The composition of these storage solutions has been designed to pro-tect the cells of the organ against hypothermic swelling, augmentenergy production following reperfusion and ablate pathologicaldamage by reactive oxygen species formed during reperfusion.2,3

Another strategy that has been shown to confer protection oncardiac tissue in a number of infarct models has been ‘ischaemicpreconditioning’ (IP), a process whereby the heart is exposed to ashort cycle of ischaemia and reperfusion (usually 2–5 min each)before an extended ischaemic period.4 Protective effects have beendemonstrated experimentally in a number of animal species5 andimplicated in humans, typically after short cycles of ischaemia andreperfusion produced by some balloon angioplasty protocols.6 Themolecular basis of IP is still incompletely understood and there havebeen many and varied potential mechanisms proposed, includingrelease of adenosine7 and activation of protein kinase C (PKC).8

Recently, intense interest has arisen in the role of ATP-sensitivepotassium (KATP) channel activation in the protection of the myo-cardium against ischaemic damage.9,10 The development of a rangeof drugs to modulate the activity of this channel has revealed a targetparticularly amenable to pharmacological intervention.11 Thesepotassium channel-opening drugs have been shown to mimic theprotective effects of IP in surgically relevant models of global cardiacischaemia and cold cardioplegic arrest,12,13 as well as in protectionof isolated human atrium.14

To date, few studies have examined the impact of IP on organpreservation in models of transplantation. Recently, our laboratoryreported enhancement of donor lung preservation by IP in a singlerat lung transplantation model after 6 and 12 h hypothermicstorage.15 Karck et al.16 have shown that IP enhances the preservationof rat heart during prolonged hypothermic storage in a number ofdifferent storage solutions. However, the potential role of the potas-sium channel was not examined in these studies.

The aims of the present study were to: (i) compare the extent towhich IP and the potassium channel opener pinacidil preserve

ATP-SENSITIVE POTASSIUM CHANNEL ACTIVATION MIMICS THEPROTECTIVE EFFECT OF ISCHAEMIC PRECONDITIONING IN THE

RAT ISOLATED WORKING HEART AFTER PROLONGEDHYPOTHERMIC STORAGE

Mark Hicks,* Zu Y Du, Paul Jansz, Steven Rainer,† Phillip Spratt and Peter S Macdonald

Cardiopulmonary Transplant Unit, *Division of Clinical Pharmacology and Toxicology and †Department of Anatomical Pathology, St Vincent’s Hospital, Darlinghurst, New South Wales, Australia

Correspondence: Dr Mark Hicks, Division of Clinical Pharmacology andToxicology, St Vincent’s Hospital, Victoria Street, Darlinghurst, NSW 2010,Australia. Email: <pmacdonald@stvincents.com.au>

Received 3 June 1998; revision 26 August 1998; accepted 31 August 1998.

Clinical and Experimental Pharmacology and Physiology (1999) 26, 20–25

IP and heart preservation 21

haemodynamic function in the rat isolated working heart model afterprolonged hypothermic storage; and (ii) whether the protectiveeffects of IP and pinacidil could be modified by pretreatment ofhearts with the potassium channel blocker glibenclamide.

METHODS

Materials and animals

Pinacidil was obtained from Eli Lilly and Co. (Indianapolis, IN, USA).Glibenclamide was purchased from Hoechst Australia Ltd (Nedlands, WA,Australia). All other chemicals were of at least AR grade and were obtainedfrom Ajax Chemical Company (Sydney, NSW, Australia).

Solution preparation

The composition of the Krebs’ solution used for organ perfusion was asfollows (in mmol/L): NaCl 118.0; KCl 4.7; MgSO4 1.2; K2HPO4 1.2; EDTA(disodium salt) 0.5; NaHCO3 25.0; CaCl2 2.5; glucose 11.0. The componentswere dissolved in water purified through a MilliQ water purification system(Millipore, Sydney, NSW, Australia). The final pH was adjusted to 7.4 withdilute H3PO4. The resultant solution was passed through a 5 mm filter andwas then bubbled continuously with a mixture of 95% oxygen and 5% carbon dioxide at 37°C. The composition of the extracellular-based cardio-plegic solution was as follows (in mmol/L): Na1 149; K1 19; Mg21 19; Ca21

2; Cl2 120; bicarbonate 28; glucose 39.2; lactate 25; aspartate 24. Pinacidilwas dissolved in dimethyl sulphoxide (DMSO) and was then diluted with

Krebs’ solution (to a final concentration of 200 mmol/L). Glibenclamide wasdissolved in Krebs’ solution (to a final concentration of 10 mmol/L).

Animals used in the study were male Wistar rats (250–330 g bodyweight),obtained from Culas Ltd (Little Bay, NSW, Australia). The study wasapproved by the Animal Ethics Committee of the Garvan Institute (Sydney,NSW, Australia). All animals received humane care in compliance with theguidelines set down by the National Health and Medical Research Councilof Australia and the ‘Principles of Laboratory Animal Care’, NationalInstitutes of Health (USA).

Experimental design

The techniques for preparing and measuring the function of the rat isolatedworking heart have been reported before by our laboratory.17,18 Isolated work-ing hearts were obtained by a technique based on that of Choong and Gavin.19

Rats were anaesthetized with halothane in 100% oxygen via a face mask. Abolus of 1000 U heparin was injected intravenously. The heart was rapidlyexcised and arrested by immersing it in 100 mL cold (2–3°C) perfusion buffer.The aorta was then ligated to a cannula and immediately perfused retro-gradely on a Langendorff perfusion apparatus with oxygenated Krebs’ per-fusion buffer at a hydrostatic pressure of 1000 mmH2O at 37°C. The leftatrium was then cannulated. This non-working preparation was run for 10 minand was then converted to a working system by switching the supply of per-fusate from the aorta to the left atrial cannula at a hydrostatic pressure of150 mmH2O. The working heart ejected perfusate via the aortic valve intothe aortic cannula. The hydrostatic pressure in the aortic cannula was main-tained at 1000 mmH2O throughout the working phase.

Hearts were allowed to stabilize for 1 min, then the following baseline in-

Fig. 1 Experimental protocols. (i) Untreated control; (ii) ischaemic preconditioning (IP); (iii) pinacidil pretreatment; (iv) dimethylsulphoxide (DMSO;pinacidil vehicle) pretreatment; (v) ischaemic preconditioning preceded by glibenclamide treatment; (vi) pretreatment with glibenclamide perfusion followedby pinacidil perfusion. (j), Langendorff perfusion; (h), aerobic perfusion in working mode; ( ), IP (5 min ischaemia); ( ), IP (5 min reperfusion); ( ),200 mmol/L pinacidil perfusion; ( ), perfusion with pinacidil vehicle (DMSO); ( ), 10 mmol/L glibenclamide perfusion; (–––), 6 or 12 h hypothermicstorage. Pre- and postischaemic functional measurements were performed 1min after conversion to working mode (arrows). Immediately before hypothermicstorage, hearts were perfused with cardioplegic solution (4°C) for 3 min.

22 M Hicks et al.

dices of cardiac function were recorded over the next 1 min period: heartrate (HR), aortic flow, coronary flow and cardiac output (CO). Aortic pres-sure was monitored in the side arm of the aortic cannula with a pressuretransducer (Ohmeda Pty Ltd, Singapore) coupled to a computerizedrecording system (Maclab, version 4; Analog Digital Instruments Pty Ltd,Sydney, NSW, Australia). The HR was derived from the pressure trace. Theaortic blood flow was measured by an inline flowmeter (TransonicsInstruments Inc., Sydney, NSW, Australia) that was also coupled to the computerized recording system. Coronary flow rate was measured by timedcollection of effluent draining from the apex of the heart. The CO was cal-culated from the sum of the aortic and coronary flow rates.

Treatment groups

Treatment groups (n 5 6 animals per group) and the time courses of treat-ment are shown in Fig. 1. The untreated control (group I) received 33 minof aerobic perfusion (10 min Langendorff perfusion followed by 23 min in‘working mode’) before cardioplegic solution (at 2–3°C) was infused intothe coronary circulation for 3 min from a reservoir located 200 mm abovethe heart. Hearts were then stored in 100 mL of the same cardioplegic solutionfor either 6 or 12 h at (2–3°C). Group II received 5 min global ischaemiafollowed by 5 min reperfusion (IP) before cardioplegia and hypothermicstorage. Group III hearts were perfused with 200 mmol/L pinacidil for 3 minbefore cardioplegia and hypothermic storage. In group IV, the pinacidilvehicle (0.1% DMSO) was substituted for pinacidil. Hearts from group Vwere perfused with 10 mmol/L glibenclamide for 3 min before IP. In groupVI, hearts were perfused with 10 mmol/L glibenclamide then 200 mmol/Lpinacidil for 3 min each.

After the 6 or 12 h storage period, each heart was remounted on the per-fusion apparatus, reperfused with oxygenated Krebs’ solution at 37°C andindices of cardiac function were remeasured, as described for baseline haemo-dynamic measurements. Hearts were then removed from the perfusion appar-atus for the determination of water content. The wet weight of a segment ofheart tissue was determined immediately, then the dry weight was determinedafter the tissue was dried in an oven at 80°C for 24 h. Water content (WC)was then calculated using the following formula:

WC 5 (wet weight (g) – dry weight (g))/dry weight (g)

Statistical analysis

All data are expressed as the mean6SEM. Differences in responses betweencontrol and treated groups were tested using two-way analysis of variance,

the factors being storage time and treatment. Post hoc comparisons were performed using Fisher’s protected least significant difference (PLSD) test(Statview 4.51; Abacus Concepts Inc., Berkeley, CA, USA). Differences wereconsidered significant if P < 0.05.

RESULTS

Pre-arrest haemodynamic measurements

The pre-arrest haemodynamic measurements are shown in Table 1.There were no significant differences between any of the treatmentsfor any of the measured variables.

Haemodynamic measurements after hypothermicstorage

Post-storage recovery of HR in the treatment groups is shown inFig. 2. After 6 h hypothermic storage, HR recovered to 8864% ofthe pre-arrest value for both the untreated control and the IP groups.The values obtained for the pinacidil and the pinacidil/glibenclamidetreatments were slightly less, although not significantly differentfrom control levels. Pretreatment of hearts with glibenclamide be-fore IP significantly decreased HR compared with control levels.After 12 h hypothermic storage, recovery of untreated control heartswas poor (1364% of the pre-arrest level). However, IP or pretreat-ment with pinacidil maintained respectable functionality (6664 and6968%, respectively). Pretreatment of hearts with glibenclamideeither before the IP cycle or pinacidil perfusion reduced recovery

Table 1 Pre-ischaemic baseline data on haemodynamic function

Group Coronary (treatment, Aortic flow flow CO HRstorage time) (mL/min) (mL/min) (mL/min) (b.p.m.)

Control6 h 5564 20.060.5 7564 27161112 h 4665 19.460.7 6666 28468

DMSO 12 h 4566 18.461.1 6567 287616

Ischaemic preconditioning6 h 5767 19.660.4 6664 31061412 h 4265 19.860.3 6265 284613

Pinacidil6 h 5464 20.060.5 7464 29661812 h 4664 21.060.3 6864 257621

GlibenclamideIP, 6 h 4264 19.860.4 6263 257615IP, 12 h 4967 21.060.3 7167 258620Pinacidil, 6 h 4064 18.061 5865 267618Pinacidil, 12 h 4764 18.461.0 6564 278616

CO, cardiac output; HR, heart rate; DMSO, dimethyl sulphoxide; IP,ischaemic preconditioning.

Fig. 2 Postischaemic recovery of heart rate (HR) after 6 (a) and 12 h (b)hypothermic storage. Values are the percentage of baseline data (Table 1).*P < 0.01 compared with untreated controls at each storage time. (n 5 6animals in each group). (h), untreated control; ( ), ischaemic precon-ditioning; (j), glibenclamide (10 mmol/L) perfusion before ischaemic pre-conditioning; ( ), pinacidil (200 mmol/L) pretreatment; ( ), glibenclamide(10 mmol/L) and pinacidil (200 mmol/L) pretreatment; ( ), dimethyl-sulphoxide (0.1%; pinacidil vehicle).

IP and heart preservation 23

to untreated control levels. The pinacidil vehicle (DMSO) was with-out protective effect on HR or any of the subsequent haemodynamicparameters measured.

Data for post-storage coronary flow are shown in Fig. 3. After 6 hhypothermic storage, flow in the IP group was maintained at 8964%of the pre-arrest value, a significant improvement over the untreatedcontrol level (6465%). All other treatments were not significantlydifferent from untreated control values. However, after 12 h storage,IP and pretreatment with the potassium channel opener again main-tained significant flow (6965 and 6563% of the pre-ischaemic levelsfor IP and pinacidil, respectively). Again, pretreatment of hearts withglibenclamide reduced flows to the level of the untreated control.

Figure 4 shows recovery of aortic flow after hypothermic storage.Recovery was poor in untreated controls (1362 and 961% of pre-arrest values for 6 and 12 h hypothermic storage, respectively). Therecovery of groups pretreated with glibenclamide, whether at 6 or12 h postarrest, was equally poor, as was the DMSO group.Recovery of flow in hearts receiving a cycle of IP or pretreatmentwith pinacidil perfusion was also incomplete, but was significantlygreater than controls or glibenclamide pretreatment (6166 and3968% of pre-arrest values for IP groups after 6 and 12 h hypo-thermic storage, respectively; 5466 and 3669% for pinacidiltreatments after 6 and 12 h hypothermic storage, respectively).

Postischaemic recovery of CO (Fig. 5) was incomplete in un-treated controls, being 2662% of pre-arrest levels after 6 h hypo-thermic storage and 1466% of pre-arrest levels for the 12 hhypothermic storage group. The recovery of hearts in the treatmentgroups containing glibenclamide was also universally poor.However, the groups exposed to IP or pinacidil alone had signifi-cantly superior CO than the aforementioned treatments after bothperiods of hypothermic storage (7164 and 5065% of pre-arrestlevels for IP groups at 6 and 12 h storage, respectively; 5764 and42.565% of pre-arrest values for groups perfused with pinacidilalone after 6 and 12 h hypothermic storage, respectively).

Fig. 3 Postischaemic recovery of coronary flow after 6 (a) and 12 h (b)hypothermic storage. Values are the percentage of baseline data (Table 1).*P < 0.01 compared with untreated controls at each storage time (n 5 6animals in each group). (h), untreated control; ( ), ischaemic precon-ditioning; (j), glibenclamide (10 mmol/L) perfusion before ischaemicpreconditioning; ( ), pinacidil (200 mmol/L) pretreatment; ( ), gliben-clamide (10 mmol/L) and pinacidil (200 mmol/L) pretreatment; ( ), di-methylsulphoxide (0.1%; pinacidil vehicle).

Fig. 4 Postischaemic recovery of aortic flow after 6 (a) and 12 h (b)hypothermic storage. Values are the percentage of baseline data (Table 1).*P < 0.01 compared with untreated controls at each storage time andglibenclamide-containing treatments at 6 h (n 5 6 animals in each group).(h), untreated control; ( ), ischaemic preconditioning; (j), glibenclamide(10 mmol/L) perfusion before ischaemic preconditioning; ( ), pinacidil(200 mmol/L) pretreatment; ( ), glibenclamide (10 mmol/L) and pinacidil(200 mmol/L) pretreatment; ( ), dimethylsulphoxide (0.1%;pinacidil vehicle).

Fig. 5 Postischaemic recovery of cardiac output (CO) after 6 (a) and 12 h(b) hypothermic storage. Values are the percentage of baseline data (Table1). *P < 0.01 compared with untreated controls at each storage time (n 5 6animals in each group). (h), untreated control; ( ), ischaemic precon-ditioning; (j), glibenclamide (10 mmol/L) perfusion before ischaemicpreconditioning; ( ), pinacidil (200 mmol/L) pretreatment; ( ), gliben-clamide (10 mmol/L) 1 pinacidil (200 mmol/L) pretreatment; ( ), di-methylsulphoxide (0.1%; pinacidil vehicle).

24 M Hicks et al.

Water content after hypothermic storage

No significant differences in WC were observed between any of thetreatment groups.

DISCUSSION

In the present study, we have shown that a single cycle of IP (5 minischaemia followed by 5 min reperfusion) will preserve haemo-dynamic function in the rat isolated working heart model after 6 and12 h of hypothermic ischaemia. These results are consistent withthose of Karck et al.,16 who demonstrated that IP maintained haemo-dynamic function in rat hearts maintained at 4°C for 10 h in a rangeof different storage solutions. We have extended this work by mim-icking the ‘ischaemic’preconditioning with a ‘pharmacological’ pre-conditioning strategy consisting of a 3 min perfusion with theATP-sensitive potassium channel opener pinacidil. Complete inhi-bition of the protection afforded by both modalities was achievedby pretreatment of the isolated hearts with the potassium channelblocker glibenclamide. This highlights the central protective role ofKATP channel activation in the context of long periods of hypothermic(ischaemic) storage periods as well as ‘classical’ IP.4,5

Although the beneficial effects of IP against ischaemia–reperfusiondamage in the majority of experimental models are clear, the exactmechanism by which IP exerts its protective effects is still unre-solved.20 In the heart and other tissues, activation and/or translocationof specific isoforms of PKC and phosphorylation of a range of ef-fector molecules are important common events.21–23 ATP-sensitivepotassium channels have been shown to be an important target forsuch kinase-mediated phosphorylation in the heart.20 Protein kinaseC has been shown to activate these channels in rabbit and humancardiac myocytes under conditions that approximate the precon-ditioning process.24,25 This ‘classical’ or immediate protection pro-vided by IP wanes after approximately 2–3 h under normothermicconditions and is followed by a further period of protectionapproximately 24 h after the initial episode of IP. It is unlikely thatdelayed protection is operative in our study, as the severe hypo-thermia would slow the synthesis of the heat shock proteins orendogenous antioxidant enzymes thought to be important in thisprocess.26 The probable explanation for the extended period of pro-tection in the study described here derives from the fact that themechanisms that cause protection to wane are probably alsotemperature dependent and will be slower under hypothermicconditions.

Cardioplegia of rat hearts with extracellular-based (St Thomas’)solution or their exposure to a single cycle of IP provides significantprotection of postischaemic pump and vascular functions after shortperiods (35 min) of normothermic global ischaemia.27 In contrast,the present study shows that hearts perfused with a similar extra-cellular-based cardioplegic solution only had profoundly impairedcoronary blood flow after 12 h hypothermic storage. However, heartspretreated with pinacidil or treated with a single cycle of IP beforecardioplegia and long-term hypothermic storage retained 65–70%of pre-ischaemic levels. This indicates significant conservation ofvascular tone in IP and pinacidil treatments. It is well known thatduring extended periods of ischaemia, endothelial cells rapidly losethe ability to synthesize nitric oxide (NO).28 Ischaemic precon-ditioning has been proposed to increase the bioavailability of NOin intestinal ischaemia/reperfusion.20,29 Production of inducible NO

synthase has been implicated in the adaptive response in the ‘secondwindow’ protective effect of IP in myocardial tissue.26 However, noassociation has yet been observed between increased flux of NO andclassical or ‘first window’ myocardial protection induced by IP.30

Potassium channel opening agents such as pinacidil are potent vaso-dilators and can markedly reduce vascular resistance.31 The increasedcoronary flow in the IP group may be a reflection of endogenousactivation of KATP channels.

Hearts stored under control conditions demonstrated a significantdepression of left ventricular function as evidenced by the very poorrecovery of aortic flow. This was presumably due to direct preser-vation injury to the cardiac myocytes. A substantial, although in-complete, improvement in pump function was observed in both IPand pinacidil pretreatments. It is not possible to judge from our studythe extent to which the improvement of cardiac function bypinacidil and IP was due to preservation of coronary blood flowversus a direct effect on the myocardium; however, because KATP

channels are distributed in high densities over both vascular endo-thelial cells and cardiac myocytes,32 it seems likely that both areimportant. The incomplete recovery of cardiac function in thepinacidil-treated group may also reflect the limited ability of the drugto homogeneously diffuse throughout the whole of the populationof cardiac myocytes during the 3 min perfusion before hypothermicstorage. Several studies reported by Grover33 imply that potassiumchannel openers must enter a slowly equilibrating compartmentbefore optimal protection is achieved, suggesting a possible intra-cellular site of action for these drugs. Such a candidate would bethe mitochondrion. It has recently been reported that severalbenzopyranyl potassium channel-opening drugs activated KATP

channels reconstituted into liposomes at cardioprotective concen-trations.34

The use of IP in the clinical context is a contentious issue. In aninitial study, patients undergoing coronary artery bypass grafting(CABG) were subjected to two cycles of 3 min of aortic crossclamping followed by 2 min reperfusion before a period of 10 minof ventricular fibrillation at room temperature, at which time distalanastomoses were made.35 Patients exposed to IP had higher levelsof cardiac ATP. Two further studies36,37 showed that when warmblood cardioplegia was used before CABG, IP produced no obser-vable improvement in outcome. Indeed, preconditioning tended toproduce a larger amount of tissue damage, as measured by increasesin creatine kinase and troponin T release, in the first 2 days afteraortic cross clamping.37 Two contexts in which IP may be usefulmay be when the risk of infarction is increased (as in the senescentmyocardium)38 and in cardiac transplantation.39 However, a moresensible approach may be to use one or a combination of pharmaco-logical approaches to mimic IP. In this manner, we have recentlydemonstrated that rat isolated working hearts recover 80% of theirpre-ischaemic CO after 12 h hypothermic storage when pretreatedwith pinacidil and stored in an extracellular-based cardioplegicsolution supplemented with the antioxidant lazaroid (U74500A).18

Although the results of the present study are promising, there area number of limitations to these findings that necessitate caution inextrapolating them to clinical heart transplantation. The results wereobtained in an in vitro system with the rat heart being perfused withphysiological saline rather than whole blood. The donor heart wasremoved from a healthy rat under general anaesthesia. Brain deathwas not induced prior to cardiectomy and the impact of brain deathon the beneficial effects of ischaemic or ‘pharmacological’ precon-

IP and heart preservation 25

ditioning is unknown. Thus, it is essential to confirm our findingsin a clinically relevant model of organ donation before any attemptis made to apply this strategy to clinical heart transplantation, a taskthat is currently being undertaken in our laboratory. However, theuse of such ‘pharmacological preconditioning’of the heart may even-tually provide a clinically useful strategy in organ retrieval in thefuture.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge funding from the National HeartFoundation of Australia and the St Vincent’s Clinic Foundation.

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