A comparative study of the most widely used solutions for cardiac graft preservation during hypothermia

A Comparative Study of the Most Widely Used Solutions for Cardiac Graft Preservation During Hypothermia Pierre Michel, PhD candidate,a Remi Vial, MD,b Claire Rodriguez, MD,c and Rene Ferrera, PhDa Background: Reports conflict on the benefits of preservative solutions. We investigated the efficacy of the most widely used cardioplegic solutions by comparing extracellular solutions such as Celsior solution, St. Thomas Hospital solutions 1 and 2 (STH-1, STH2), the modified University of Wisconsin solution (UW-1), Lyon Preservation solution (LYPS) from our laboratory, and intracellular solutions such as standard University of Wisconsin solution (UW), Bretschneider solution (HTK), Stanford solution (STF), and Euro-Collins solution (EC). Methods: Male rats (n ⫽ 110) were randomized into 11 groups: LYPS, Celsior, STH-1, STH-2, UW-1, UW, HTK, STF, EC, and normal saline solution groups, and a control group. All hearts, except controls, were preserved by cold storage (8 hours at 4°C) in the various solutions. We used an isolated non–working-heart model and biopsy specimens to assess heart preservation (n ⫽ 5/group). Results: Hearts stored in the EC and saline solutions had poor left ventricular developed pressure (LVDP) ⫻ heart rate (HR) (1,407.5 ⫾ 154 and 1,390 ⫾ 439 mm Hg/mn, respectively). In contrast, hearts stored in LYPS and Celsior had a LVDP ⫻ HR close to control hearts (31,349 ⫾ 1,847, 27,620 ⫾ 1,207, and 36,627 ⫾ 1,322 mm Hg/mn, respectively), whereas hearts stored in STH-1, STH-2, UW-1, UW, HTK, and STF had intermediate functional response (14,278 ⫾ 2,176, 12,402 ⫾ 1,571, 11,428 ⫾ 1,629, 11,603 ⫾ 2,521, 7,045 ⫾ 537, and 7,086 ⫾ 1,206 mm Hg/mn, respectively). Hearts preserved with STH-2, UW, HTK, STF, EC, and saline solution showed significantly increased release of creatine kinase and lactate dehydrogenase than did control hearts or hearts preserved in Celsior, LYPS, STH-1, and UW-1. The energetic charge (EC ⫽ [(0.5 adenosine diphosphate ⫹ adenosine triphosphate) ⫼ (adenosine triphosphate ⫹ adenosine diphosphate ⫹ adenosine monophosphate)]) in STH-2, UW, HTK, STF, EC, and saline groups was significantly lower (p ⬍ 0.05) than in the other groups. Conclusion: Extracellular-type solutions provided better preservation than did intracellular-type solutions. However, UW and UW-1 (intracellular- and extracellulartype solutions) provided equivalent preservation of cardiac function. Preservation quality may be attributed to calcium, often added to extracellular solutions. Among extracellular solutions, Celsior and LYPS solution showed comparable efficacy on left

From the aInstitut Fe´de´ratif de Recherche Cardiovasculaire, b Ho ˆpital Edouard Herriot, and cHo ˆpital Pneumologique et Cardiovasculaire Louis Pradel, Lyon, France. Submitted September 6, 2001; revised October 24, 2001; accepted January 8, 2002. Reprint requests: Rene´ Ferrera, Universite´ Claude BernardLyon I Laboratoire de Physiologie, Faculte´ de Me´decine Lyon

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Nord 4e´me ´etage-Escalier C ou D, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France. E-mail: [email protected] Copyright © 2002 by the International Society for Heart and Lung Transplantation. 1053-2498/02/$–see front matter

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ventricular function and seemed to offer better preservation than the other solutions tested in this study. J Heart Lung Transplant 2002;21:1030 –1039.

L

imited preservation time of the adult donor heart, usually less than 4 to 6 hours, presents a practical problem in heart transplantation. Longer preservation time is necessary to increase the donor pool and to provide adequate time for graft assessment. To reach this goal, several solutions for preserving the cardiac graft have been proposed and evaluated experimentally and clinically. St. Thomas Hospital solutions (STH-1 and STH-2) were introduced in clinical practice in 1975 and 1981, respectively. University of Wisconsin (UW) solution and Euro-Collins (EC) solution were formulated as universal preservation solutions and tested for cardiac preservation after excellent results with kidney, liver, and pancreas preservation.1–5 Histidine-tryptophane-ketoglutarate solution (HTK) was initially developed as a cardioplegic solution by Bretschneider et al.6 Controversy persists regarding selection of heart preservation solution. According to Choong et al,7 STH-2 solution is superior to UW solution for cardiac preservation, whereas Ledingham et al8 reported that UW solution was better. However, Demertzis et al9 concluded that these solutions were comparable when mean ischemic time was ⬍4 hours. The superiority of HTK solution vs UW solution was also discussed. According to Ku et al,10 HTK solution is much more effective than UW solution, whereas Galinanes et al,11 found UW solution marginally superior to HTK solution. Swanson et al12 evaluated UW, modified EC, and Stanford (STF) solutions with an isolated working canine heart model and obtained superior preservation with UW after 12 hours of cold storage. More recently Celsior was introduced in clinical practice. Before clinical use, Celsior and STH-2 solutions were tested in an isolated rat heart model and a heterotopic rabbit heart transplantation model. According to Menasche et al,13 Celsior is superior to STH-2 solution for preserving the myocardium. In summary, more than 10 solutions, some similar and some very different in composition, are used in the clinical setting. Regarding sodium-to-potassium ratio, cold storage solutions can be divided into 2 categories: intracellular and extracellular preservation types. The choice of electrolyte ratio is also discussed. Inversion of the sodium-to-potassium ratio in UW (extracellular type solution) seems to induce notable improvement in preserving hearts,14

whereas Ko et al15 recommend using the intracellular electrolyte composition. In our study we compared the efficacy of several cardioplegic solutions by comparing extracellulartype solutions such as LYPS, Celsior, STH-1, STH-2, UW-1, and intracellular-type solutions such as UW, HTK, STF, and EC. To evaluate these solutions, we used an isolated non–working-heart model. We assessed the quality of myocardial preservation using cardiac functional indices, changes in the level of adenylic nucleotides, and enzymatic release.

MATERIAL AND METHODS Humane Animal Care All animals received humane care in accordance with the Principles of Laboratory Animal Care, formulated by the national Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86 –23, revised 1985)

Experimental Procedure Wistar male rats, weighting 350 to 450 g, were randomized in 10 groups, corresponding to the 10 solutions tested (Table I) : LYPS, Celsior, STH-1, STH-2, UW-1, UW, HTK, STF, EC, and normal saline solution. In the control group, hearts were harvested and immediately reperfused. Rats were anesthetized with pentobarbital (50 mg/kg), and heparin (200 UI/kg) was injected into the femoral vein. The heart was rapidly excised and immersed in 1 of the 10 preservation solutions (4°C). The aorta was cannulated and within 1 minute, and the cardiac coronary vessels were rinsed retrogradely with the same solution for 1 minute at a constant perfusion pressure (100 cm H2O). We paid special attention to achieving strictly homogeneous initial conditions for all hearts before applying cold retrograde perfusion. The hearts, except controls, were preserved by simple immersion for 8 hours at 4°C in 1 of the various solutions (n ⫽ 10/group).

Functional Evaluation of Preserved Hearts At the end of the storage period, the hearts were immediately frozen in liquid nitrogen (n ⫽ 5/group) or perfused retrogradely in Langendorff mode (n ⫽

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TABLE I Composition of the preservation solutions Components

LYPS

Celsior

STH-1

STH-2

UW-1

UW

HTK

STF

EC

NaCl

Type Electrolytes Na⫹ K⫹ Ca2⫹ Cl⫺ Mg2⫹ SO42⫺ HPO43⫺ H2PO42⫺ HCO3⫺ Metabolic agents Glucose Aspartate Glutamate ␣-Ketoglutarate Tryptophan Insulin (UI/liter) Pyruvate Adenosine Impermeants Lactobionate Mannitol PEG D⫹ raffinose HES (g/liter) Antioxidants Allopurinol Reduced glutathione Buffers Histidine Histidine-HCl HEPES Miscellaneous Chlorpromazine-HCl Procaine-HCl

Extra

Extra

Extra

Extra

Extra

Intra

Intra

Intra

Intra

Extra

110 20 1 150 4 — — — —

100 15 0.25 41.5 13 — — — —

147 20 2 203 16 — — — —

120 16 1.2 160 16 — — — 10

125 30 — — 5 5 — 25 —

30 125 — — 5 5 — 25 —

15 9 0.015 32.03 4 — — — —

20 27 — 27 — — — — 20

15 115 — 15 — — 42 15 15

154 — — 154 — — — — —

20 2 1.4 — — 250 2.5 —

— — 20 — — — — —

— — — — — — — —

— — — — — — — —

— — — — — — — 5

— — — — — — — 5

— — — 1 2 — — —

250 — — — — — — —

194 — — — — — — —

— — — — — — — —

— — 2 — —

80 60 — — —

— — — — —

— — — — —

100 — — 35.36 50

100 — — 35.36 50

— 30 — — —

— 60 — — —

— — — — —

— — — — —

— 0.3

— 3

— —

— —

1 3

1 3

— —

— —

— —

— —

— — 20

30 — —

— — —

— — —

— — —

— — —

180 18 —

— — —

— — —

— — —

0.7 —

— —

— 1

— 1

— —

— —

— —

— —

— —

— —

Concentrations are given as millimoles per liter (mmol/liter) unless otherwise noted. LYPS, Lyon preservative solution; STH-1, St. Thomas Hospital 1 cardioplegic solution; STH-2, St. Thomas Hospital 1 cardioplegic solution; UW-1, University of Wisconsin modified solution; UW, Standard University of Wisconsin; HTK, Bretschneider solution; STF, Stanford solution; EC, Euro-Collins solution; Extra, extracellular-type solution; Intra, intracellular-type solution; PEG, polyethyleneglycol; HES, hydroxyethyl starch.

5/group).16 Reperfusion lasted 60 minutes at 37°C under a hydrostatic pressure of 100 cm H2O using Krebs-Henseleit bicarbonate buffer (containing 11.0 mmol/liter glucose, 118.5 mmol/liter NaCl, 4.75 mmol/liter KCl, 1,19 mmol/liter MgSO4, 1.18 mmol/ liter KH2PO4, 25.0 mmol/liter NaHCO3, 1.4 mmol/ liter CaCl2 with a pH of 7.4 and continuously bubbled with 95% O2 and 5% CO2). Left ventricular pacing at a constant rate of 300 beats/min was established. We determined coronary flow through a timed series of collections of coronary

effluent. We measured the left ventricular systolic pressure (LVSP) and the left ventricular end-diastolic pressure (LVEDP) using a latex balloon introduced in the left ventricle and expanded to exert a physiologic end-diastolic pressure of 5 mm Hg. We calculated the functional index (LVSP ⫺ LVEDP) ⫻ HR or LVDP ⫻ HR. Contractility was calculated as the maximum rate increase in the pressure curve or LV dP/dt max, and the maximum isovolumetric rate of relaxation was calculated from the rate decrease of the pressure curve or LV dP/dt min.

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FIGURE 1 Functional assessment of hearts during reperfusion. Effects of the Lyon

preservative solution (LYPS), Celsior, St. Thomas Hospital 1 and 2 (STH-1, STH-2), modified University of Wisconsin (UW-1), standard University of Wisconsin (UW), Bretschneider (HTK), Stanford (STF), Euro-Collins (EC) and saline solutions on the functional index LVDP ⫻ HR during 60 minutes of reperfusion. The control hearts were excised and immediately reperfused. The values shown are the mean of 5 values ⫾ SEM. *p ⬍ 0.05, ***p ⬍ 0.001, different for the control group value; }}}p ⬍ 0.001, different for the LYPS group value; †††p ⬍ 0.001, different for the Celsior group value; ■■p ⬍ 0.01, ■■■ p ⬍ 0.001, different for the STH-1 group value; 䊐䊐䊐p ⬍ 0.001, different for the STH-2 group value; FFFp ⬍ 0.001, different for the UW-1 group value; EEEp ⬍ 0.001, different for the UW group value; ✛p ⬍ 0.05, different for the HTK group value; p ⬍ 0.05, different for the STF group value. LVDP, left ventricular developed pressure; HR, heart rate.

Evaluation of the Cellular Damage

Statistical Analyses

Creatine kinase and lactate dehydrogenase release were determined in the coronary effluent at the end of the reperfusion using a validated dosage kit (Merck KGaA, 64271; Darmstadt, Germany).

We performed statistical comparisons using the analysis of variance Fisher PLSD test, which permitted comparison of all groups simultaneously. Each group contained 5 hearts. All results are expressed for each group as the mean ⫾ the standard error of the mean (SEM). Limits of significance are p ⫽ 0.05, p ⫽ 0.01, and p ⫽ 0.001.

Energy Evaluation of Preserved Hearts Ventricular biopsy specimens of the hearts were frozen at the end of the cold storage period. Adenine nucleotides (adenosine monophosphate [AMP], adenosine diphosphate [ADP], and adenosine triphosphate [ATP]) and the catabolites (hypoxanthine and xanthine) were separated by highperformance liquid chromatography, as described by Hull-Ryde et al.17 The sum of adenylic nucleotides (⌺ AN ⫽ ATP ⫹ ADP ⫹ AMP) and the energetic charge EC ⫽ [(0.5 ADP ⫹ ATP) / ⌺ AN] were calculated.

RESULTS Functional Evaluation of Reperfusion Figure 1 shows the recovery of cardiac function index LVDP ⫻ HR after 8 hours of cold storage. It can be distinguished by 3 sub-groups. The first is the EC and saline hearts, which had poor functional recovery during reperfusion (1,407.5 ⫾ 154 and 1,390 ⫾ 439 mm Hg/mn, respectively). In addition, saline hearts were very arrhythmic, despite sustained electric stim-

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FIGURE 2 Effects of the Lyon preservative solution (LYPS), Celsior, St.Thomas Hospital

1 and 2 (STH-1, STH-2), modified University of Wisconsin (UW-1), standard University of Wisconsin (UW), Bretschneider (HTK), Stanford (STF), Euro-Collins (EC) and saline solutions on recovery of contractility (LV dP/dt max) and the maximum isovolumetric rate of relaxation of the left ventricle (LV dP/dt min) during reperfusion. The values shown are the mean of 5 values ⫾ SEM. ***p ⬍ 0.001, different for the control group value; }}}p ⬍ 0.001, different for the LYPS group value; †††p ⬍ 0.001, different for the Celsior group value; ■■p ⬍ 0.01, ■■■p ⬍ 0.001, different for the STH-1 group value; 䊐䊐䊐p ⬍ 0.001, different for the STH-2 group value; FFp ⬍ 0.01, FFFp ⬍ 0.001, different for the UW-1 group value; EEp ⬍ 0.01, EEEp ⬍ 0.001, different for the UW group value; ✛p ⬍ 0.05, different for the HTK group value; p ⬍ 0.05, different for the STF group value.

ulation at a constant frequency of 300 beats/min. The second sub-group consisted of hearts preserved in STH-1, STH-2, UW-1, UW, HTK, and STF (14,278 ⫾ 2,176, 12,402 ⫾ 1,571, 11,428 ⫾ 1,629, 11,603 ⫾ 2,521, 7,045 ⫾ 537 and 7,086 ⫾ 1,206 mm Hg/mn, respectively). In this second group, hearts showed intermediate functional recovery. Hearts preserved with LYPS and Celsior solutions formed the third group, with cardiac function indices similar to the control hearts (31,349 ⫾ 1,847, 27,620 ⫾ 1,207 and 36,627 ⫾ 1,322 mm Hg/mn, respectively). The recovery of contractility (LV dP/dt max) and the maximum isovolumetric rate of relaxation of the left ventricle (LV dP/dt min) were significantly better in control hearts than in preserved hearts (p ⬍ 0.001) (Figure 2). Among the preserved hearts, those preserved with LYPS and Celsior had the best functional recovery. The coronary flow of hearts preserved with LYPS, Celsior, STH-2, UW-1, UW, HTK, STF, EC, and NaCl was not significantly different from that of the control hearts (13.4 ⫾ 1.5, 12.6 ⫾ 2.9, 10.7 ⫾ 0.7,

13.8 ⫾ 1.8, 13.7 ⫾ 2.2, 12.4 ⫾ 2, 8.6 ⫾ 0.5, 9.4 ⫾ 0.6, 10.7 ⫾ 0.6, and 12.5 ⫾ 0.4 ml/min/g, respectively). In the STH-1 group, the coronary flow (7.9 ⫾ 0.5 ml/min/g) was significantly lower than in hearts in the LYPS, Celsior, UW-1 (p ⬍ 0.01), and control (p ⬍ 0.05) groups.

Evaluation of Cellular Damage Figure 3 shows the release of creatine kinase and lactate dehydrogenase at the end of reperfusion. The hearts preserved with STH-2, UW, HTK, STFD, EC, and saline solutions had significantly increased creatine kinase and lactate dehydrogenase release in the coronary effluent compared with the control hearts. Release of enzymes in the LYPS, Celsior, STH-1, and UW-1 groups was similar, with no significant difference from the control group.

Preservation and Myocardial Adenine Nucleotide Levels The ATP level (Table II) and the adenylate charge (Figure 4) of preserved hearts were signif-

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FIGURE 3 Enzymatic release during reperfusion. Creatine kinase release (CK) and lactate

dehydrogenase (LDH) in the coronary effluent during the reperfusion in the control and the preserved hearts: Lyon preservative solution (LYPS), Celsior, St. Thomas Hospital 1 and 2 (STH-1, STH-2), modified University of Wisconsin (UW-1), standard University of Wisconsin (UW), Bretschneider (HTK), Stanford (STF), Euro-Collins (EC), and saline solutions. The values shown are the mean of 5 values ⫾ SEM. *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.001, different for the control group value; }}p ⬍ 0.01, }}}p ⬍ 0.001, different for the LYPS group value; †p ⬍ 0.05, ††p ⬍ 0.01, †††p ⬍ 0.001, different for the Celsior group value; ■p ⬍ 0.05, ■■p ⬍ 0.01, different for the STH-1 group value; 䊐p ⬍ 0.05, different for the STH-2 group value; Fp ⬍ 0.05, FFp ⬍ 0.01, different for the UW-1 group value; Ep ⬍ 0.05, EEp ⬍ 0.01, different for the UW group value.

icantly lower than that of the control hearts. Among the preserved groups, the Celsior group contained significantly more ATP. The LYPS, Celsior, STH-1, and UW-1 groups had better energetic charge than STH-2, UW, HTK, STF, EC, and saline groups (0.568 ⫾ 0.009, 0.65 ⫾ 0.034, 0.542 ⫾ 0.033, 0.578 ⫾ 0.027, 0.362 ⫾ 0.046, 0.142 ⫾ 0.01, 0.197 ⫾ 0.017, 0.223 ⫾ 0.029, 0.295 ⫾ 0.094, 0.351 ⫾ 0.077, respectively). With regard to the sum of adenylic nucleotides, hearts preserved with LYPS and Celsior had values slightly higher than control hearts but without significant difference. Inosine release (Table II) in the preserved hearts was more significant than in control hearts, except in the Celsior and EC groups. The hypoxanthine level was significantly higher in the LYPS, STH-2, UW-1, HTK, STF, EC and saline hearts than in the control hearts. Among the preserved groups, HTK and saline hearts released significantly more inosine and hypoxanthine, respectively.

DISCUSSION In hearts preserved for 8 hours at 4°C with saline solution, LVDP ⫻ HR, contractility, and maximum isovolumetric rate of relaxation were considerably lower than that measured in the other groups (see Figures 1 and 2). Therefore, we found simple isotonic saline solution completely insufficient to preserve hearts at 4°C for even a few hours. Several clinical teams use STH-1 cardioplegic solution to store donor hearts. It is a relatively simple solution because it contains only 5 electrolytes. Despite its simplicity, STH-1 ensured better functional, enzymatic, and energy preservation than did the saline solution. The ionic basis of the solution seems of primary importance in improving the quality of the preservation. The preserved hearts were all stored at 4°C for 8 hours. During this cold storage period, the solutions did not penetrate into the tissue and so did not seem to affect myocardial viability. Only the medium in the coronary vessels acted on endothelial

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TABLE II Energetic indexes of hearts after simple cold storage Groups

ATP

⌺ NA

Inosine

Hypoxanthine

Control LYPS Celsior STH-1 STH-2 UW-1 UW HTK STF EC NaCl

23.5 ⫾ 1.2 16.6 ⫾ 2.0b 25.2 ⫾ 4.2e 11.3 ⫾ 2.2c,d,h 6.4 ⫾ 1.21e,f,h,i,k,l 14.0 ⫾ 2.0c,h,m 1.9 ⫾ 0.25c,h,k,p 2.9 ⫾ 0.78c,f,h,k,p 2.9 ⫾ 0.57c,f,h,kp 3.7 ⫾ 1.01c,f,h,j,p 5.0 ⫾ 2.2c,f,h,i,m,o

41.8 ⫾ 1.8 53.4 ⫾ 6.0 59.6 ⫾ 5.8a 40.0 ⫾ 4.3g 43.8 ⫾ 9.9 40.2 ⫾ 3.4g 30.2 ⫾ 1.3e,h 30.8 ⫾ 4.6a,e,h 27.3 ⫾ 2.3c,f,h,l 27.5 ⫾ 5.2f,h,l 40.7 ⫾ 10.5j

0 ⫾ 0.0 7.7 ⫾ 3.1a 6.5 ⫾ 0.7 6.8 ⫾ 4.0a 10.4 ⫾ 1.16b 6.8 ⫾ 2.1a 12.3 ⫾ 0.9c 12.7 ⫾ 1.5c 12.5 ⫾ 1.3c 4.0 ⫾ 1.1q,s 15.1 ⫾ 4.8c,e,g,i,t

0.3 ⫾ 0.1 2.7 ⫾ 0.2a 2.3 ⫾ 0.1 2.0 ⫾ 0.2 3.6 ⫾ 1.2b 2.7 ⫾ 0.2a 2.0 ⫾ 0.2 4.7 ⫾ 1.2c,e,g,n,r 3.7 ⫾ 0.4b 2.5 ⫾ 0.1a 3.4 ⫾ 0.7b

Comparison of the ATP level, the sum of adenylic nucleotides (⌺ NA) and inosine and hypoxanthine, levels (nmol/g of protein) were evaluated from cardiac biopsy specimens of control and preserved hearts. The values shown are the mean of 5 values ⫾ SEM. a p ⬍ 0.05, bp ⬍ 0.01, cp ⬍ 0.001, different for the control group value; dp ⬍ 0.05; ep ⬍ 0.01, fp ⬍ 0.001, different for the LYPS group value; gp ⬍ 0.05, hp ⬍ 0.01, different for the Celsior group value; ip ⬍ 0.05, jp ⬍ 0.01, kp ⬍ 0.001, different for the STH-1 group value; l p ⬍ 0.05, mp ⬍ 0.01, different for the STH-2 group value; np ⬍ 0.05, op ⬍0.01, pp ⬍ 0.001, different fro the UW-1 group value; qp ⬍ 0.05, r p ⬍ 0.01, different for the UW group value; sp ⬍ 0.01, different for the HTK group value; tp ⬍ 0.01, different for the EC group value. ATP, adenosine triphosphate; LYPS, Lyon preservative solution; STH-1, St. Thomas Hospital 1 cardioplegic solution; STH-2, St. Thomas Hospital 1 cardioplegic solution; UW-1, University of Wisconsin modified solution; UW, Standard University of Wisconsin; HTK, Bretschneider solution; STF, Stanford solution; EC, Euro-Collins solution; Extra, extracellular-type solution; Intra, intracellular-type solution.

and cardiomyocyte cells. The hearts of the various groups were thus separated only by 1 minute of coronary wash performed at the beginning of storage. It was this minute of initial wash that induced impressive differences among groups 8 hours later. Cold storage solutions can be classified into 2 types, extracellular and intracellular, based on sodium and potassium concentrations. Extracellulartype solutions mimic extracellular fluids and have a sodium concentration ⱖ70 mmol/liter and a potassium concentration between 5 and 30 mmol/liter. Conversely, intracellular-type solutions mimic intracellular fluids have a sodium concentration ⬍70 mmol/liter and a potassium concentration ranging between 30 and 125 mmol/liter.18 The advantage of intracellular solutions is their low osmolarity, which allows adding high concentrations of impermeants and their capacity to induce rapid cardiac arrest. The major disadvantage of intracellular solutions is endothelial dysfunction, notably autoregulation impairment caused by high potassium concentrations.19 Selecting one of the 2 types of preservative solution, extracellular or intracellular, remains controversial.14,15 In this study, functional recovery (Figures 1 and 2) and enzyme release (Figure 3) with LYPS, Celsior, STH-1, and STH-2 hearts were superior to that of HTK, STF, and EC hearts. This findings supports Choong et al7 and Southard,14 who found extracellular solution superior to intracellular solution for heart preservation. However, our re-

sults indicate equivalent functional recovery with UW (intracellular) and UW-1 (extracellular) solutions. The inversion of the sodium-to-potassium ratio in UW solution had no effect on myocardial preservation, which was inconsistent with the above results. Nevertheless, our observations support those of Drinkwater et al.20 The only difference between the 2 solutions in our study was the concentrations of sodium and potassium. Although, the presence of sodium and potassium ions seem essential, a superior protective effect of intracellular or extracellular solution (based on the sodium and potassium concentrations) was not demonstrated in this study. Therefore, what was the difference in efficacy among groups? Hearts preserved with UW, UW-1, HTK, STF, EC, and NaCl solutions had inferior left ventricular function (by 30%) compared with control hearts (Figure 1). These preservative solutions lack calcium, except for HTK (0.015 mmol/liter). Moreover, the solutions that better preserve heart viability (LYPS, Celsior, STH-1, and STH-2) contained calcium at a concentration ⬎0.1 mmol/liter, which appears to be the lower limit for efficacy.21 The deleterious effect of the absence of calcium during preservation is clear. Calcium prevents calcium paradox. A previous works showed the beneficial effect of calcium supplementation in UW (1 mmol/liter).22 The results of our study support this. However, several studies have shown that decreasing calcium

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FIGURE 4 Adenylate charge of hearts after simple cold storage obtained by biopsy from

control and preserved hearts: Lyon preservative solution (LYPS), Celsior, St. Thomas Hospital 1 and 2 (STH-1, STH-2), modified University of Wisconsin (UW-1), standard University of Wisconsin (UW), Bretschneider (HTK), Stanford (STF), Euro-Collins (EC), and saline solutions. The values shown are the mean of 5 values ⫾ SEM. **p ⬍ 0.01, ***p ⬍ 0.001, different for the control group value; }}}p ⬍ 0.001, different for the LYPS group value; †††p ⬍ 0.001, different for the Celsior group value; ■■p ⬍ 0.01, ■■■p ⬍ 0.001, different for the STH-1 group value; 䊐䊐p ⬍ 0.01, 䊐p ⬍ 0.05, 䊐䊐䊐p ⬍ 0.001, different for the STH-2 group value; FFFp ⬍ 0.001, different for the UW-1 group value; EEp ⬍ 0.01, different for the UW group value; ✛p ⬍ 0.05, different for the HTK group value.

overload during ischemia–reperfusion improves myocardial recovery after cold storage.23,24 Various strategies have been proposed to prevent or reduce calcium overload during ischemia and reperfusion: (1) low concentration of calcium,25 (2) calcium channel blockers,25 (3) sodium/potassium exchange inhibitors,25 and (4) hyperpolarized arrest with potassium channel openers26 or sodium channel inhibitors.27 Celsior and LYPS are new preservative solutions that contain compounds intended to limit the deleterious effects or ischemia and reperfusion. In preliminary studies, we demonstrated the myocardial protective effects of 4 compounds (pyruvate, aspartate, glutamate, and glucose) during hypothermia and reperfusion.28,29 Aspartate and glutamate are essential for myocardial protection.30,31 Beyond its effect on pyruvate dehydrogenase activation and aerobic stimulation, pyruvate also improves recovery of heart function by preventing free-radical generation,32 limiting the deleterious effects of fatty acids.33 Glucose and insulin may increase glycolysis and limit ade-

nine nucleotides depletion.34,35 In hibernating animals such as the frog, blood glucose has been shown to reach 250 mmol/liter. We think that, in addition to its energetic effect, glucose may protect cardiac cells at low temperatures by unexplored mechanisms, perhaps acting directly on cardiomyocyte membranes, or because of the increase viscosity of the solution. We observed no relation between energy additives and adenine nucleotides levels at the end of cold storage. Indeed, the STH-1 and UW-1 groups had an energetic charge not significantly different from the Celsior and LYPS groups (Figure 4). However, Celsior and LYPS solutions contained additives intended to improve the energy stores, in contrast with the STH-1 solution that contained only electrolytes. Thus we question the need to use such compounds to limit depletion of highenergy phosphates. However, the discrepancies between cardiac function and energy levels indicated that the ATP level, the sum of catabolites, and the energetic charge probably are not sufficient indicators of the state of cardiac function

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after preservation. Our results showed only that LYPS, Celsior, STH-1, and UW-1 solutions maintained a higher myocardial energy level than did the others solutions. Moreover, LYPS and Celsior solutions contain a buffer component (HEPES and histidine, respectively). Other compounds were added to limit edema (mannitol, lactobionate or polyethyleneglycol36), to prevent free-radical production (reduced glutathione37), and to stabilize cellular membrane (chlorpromazine38). In this study, hearts preserved with LYPS and Celsior solution for 8 hours of simple cold storage had better functional assessment than hearts preserved in the other solutions (Figures 1 and 2). Indeed, LYPS and Celsior hearts had a left ventricular function (LVDP ⫻ HR) equivalent to 80% of the controls hearts. The hearts preserved in STH-1, STH-2, UW-1, UW, HTK, STF, and EC had left ventricular function approximately 40% that of the controls hearts. Therefore, LYPS and Celsior solutions had twice the protective effect on ventricular function of the other solutions.

CONCLUSION Calcium was of primary importance to improved preservation. With regard to UW and UW-1 results, intracellular and extracellular solutions provided equivalent preservation of cardiac function. Celsior and LYPS had comparable efficacy for left ventricular function. These solutions seems to offer better preservation than did the other solutions tested in this study. To extend the hypothermic period, adequate preservation solution and improved techniques are necessary. Experiments are planned to further evaluate LYPS and Celsior for hypothermic heart perfusion. The authors thank Colette Berthet for expert technical assistance.

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