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Ann Thorac Surg 1995;59:1127-1133
© 1995 The Society of Thoracic Surgeons

Cardiac Storage With University of Wisconsin Solution and a Nucleoside-Transport Blocker

Divisions of Cardiovascular Surgery and Clinical Biochemistry, Centre for Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada

Accepted for publication January 19, 1995.

	Abstract

Top Footnotes Abstract Introduction Methods Results Comment Acknowledgments References

 
Findings from previous investigations conducted at this institution and others have suggested that University of Wisconsin solution (UWS) is preferable for the prolonged hypothermic storage of hearts before transplantation. The benefit seen with UWS may in part be related to the inclusion of adenosine (5 mmol/L) in the UWS. To investigate whether further manipulations of adenosine metabolism might enhance myocardial protection, studies were initially conducted using cultured myocytes, followed by confirmatory experiments using isolated rat hearts. Cultured human ventricular myocytes (7 to 8 dishes/group) were stored for 12 hours at 0°C in unmodified UWS or UWS supplemented with increasing concentrations (1 to 100 µmol/L) of the nucleoside-transport blocker p-nitrobenzylthioinosine. The adenosine triphosphate concentrations were found to be enhanced with nucleoside-transport inhibition, with the best results achieved with the 1- and 3-µmol/L groups (control, 3.37 ± 0.41 nmol/µg DNA; UWS, 2.89 ± 1.31 nmol/µg DNA; 1 µmol/L, 5.91 ± 3.23 nmol/µg DNA; 3 µmol/L, 7.86 ± 3.45 nmol/µg DNA; p < 0.05 versus control or UWS group). Isolated rodent hearts from Sprague-Dawley rats were prepared on a Langendorff apparatus with an intraventricular balloon and subsequently stored for 8 hours at 0°C in unmodified UWS (13 hearts/group) or UWS supplemented with 1 or 3 µmol/L of p-nitrobenzylthioinosine (9 to 10 hearts/group). In separate experiments (5 to 6 hearts/group), the tissue levels of purine metabolites were monitored. The addition of the nucleoside-transport blocker was associated with an increased postischemic developed pressure (UWS, 66.2% ± 11.1%; 1 µmol/L, 75.8% ± 6.4%; 3 µmol/L, 78.3% ± 10.7%; p < 0.05 UWS versus 1- or 3-µmol/L group) while diastolic compliance was reduced after storage in all groups (p = 0.01). Coronary flow was increased in association with the nucleoside-transport blocker (UWS, 71.3% ± 16.7%; 1 µmol/L, 78.5% ± 11.0%; 3 µmol/L, 86.6% ± 13.3%; p < 0.05 UWS versus 3 µmol/L). The p-nitrobenzylthioinosine did seem to block nucleoside transport, as the hypoxanthine content was unmeasurable after perfusion in the treated groups compared with the findings in the group receiving unmodified UWS (p < 0.01).

	Introduction

Top Footnotes Abstract Introduction Methods Results Comment Acknowledgments References

 
Findings from previous investigations conducted at this institution [1, 2] and at others [3–5] recommend the use of University of Wisconsin solution (UWS) for the prolonged hypothermic storage of hearts before transplantation. University of Wisconsin solution contains the cardioprotective agent adenosine (5 mmol/L), which may contribute to the improved results [6]. The benefits of exogenous or endogenous adenosine observed in various ischemia–reperfusion models are enhanced by adenosine deaminase inhibition [7], acadesine [8], or blockers of transmembrane nucleoside transport that result in increased levels of tissue nucleosides [9]. To determine whether the addition of a nucleoside-transport blocker can augment cardiac recovery after prolonged hypothermic storage with UWS, we carried out experiments using p-nitrobenzylthioinosine (NBMPR), initially using cultured human cardiomyocytes as a screening test. These results subsequently were applied in the setting of an isolated rodent heart preparation.

	Methods

Top Footnotes Abstract Introduction Methods Results Comment Acknowledgments References

 
Human Cardiomyocyte Experiments
Cultured human ventricular cardiomyocytes were prepared from infundibular myocardium of the right ventricular outflow tract, as previously described [1, 2]. Myocardium was obtained from patients with tetralogy of Fallot, aged 2 to 4 years, whose parents had signed a consent form approved by the institutional human ethics committee. All experiments were performed using cell cultures established from a single patient. Control results were obtained by discarding the culture medium, rinsing the cells with phosphate-buffered saline solution, and then freezing the cells with liquid nitrogen. To obtain the storage results, after removal of the culture media and rinsing the cells with phosphate-buffered saline solution, 12 mL of a chilled test solution was placed in a culture dish. The dishes were stored for 12 hours at 0°C, after which the preservation fluid was discarded, the cells were rinsed with phosphate-buffered saline, and the cells were then frozen with liquid nitrogen.

The NBMPR (s-[p-nitrobenzyl]-6-thioinosine) was obtained from Sigma Chemical Company (St. Louis, MO). The storage solutions consisted of UWS or UWS supplemented with 1, 3, 10, 30, or 100 µmol/L of NBMPR (groups UWS, N1, N3, N10, N30, and N100, respectively). The NBMPR was solubilized in a vehicle composed of dimethyl sulfoxide (Anachemia, Toronto, Ontario, Canada). A stock solution with an NBMPR concentration of 20 mg/mL was prepared, and aliquots of this stock were employed to formulate the various concentrations of NBMPR in UWS, with appropriate corrections made for the final volume. There were seven to eight dishes per group. Additionally, cells were stored in UWS supplemented with the vehicle but without NBMPR.

The methods for purine extraction and analysis have been described elsewhere in extensive detail [10]. The DNA in the pellets remaining from the adenine nucleotide analysis was assayed using the method of Burton [11]. Cold (4°C) 5% perchloric acid was added to each sample, followed by mixing and incubation on ice for at least 10 minutes. After centrifugation at 12,000 g for 10 minutes, the supernatants were removed and the pellets were resuspended in cold 5% perchloric acid and kept on ice for 10 minutes. All samples and a DNA standard (1.0 mg/mL of calf thymus DNA [Sigma]) in 5 mmol/L of sodium hydroxide and an equal volume of cold 10% perchloric acid were placed in a 70°C water bath for 15 minutes. The samples and standard were then spun at 12,000 g for 10 minutes and the supernatants transferred to new tubes. A diphenylamine reagent consisting of diphenylamine (88.6 mmol/L), glacial acetic acid (98%), concentrated sulfuric acid (1.5%), and a 16-mg/mL acetaldehyde solution (0.5%) was added to each of the samples and DNA standard aliquots and to a tube containing 5% perchloric acid (blank) in a ratio of two parts of the diphenylamine reagent to one part of the sample. All of the samples and standards were then incubated at room temperature in darkness for 16 hours. The optical densities of the standards and samples were then measured on a spectrophotometer (DU-40; Beckman Instruments, Irvine, CA) at a spectral wave length of 600 nm. The concentrations of adenine nucleotides and their degradation products were expressed as nanomoles per microgram of DNA.

Isolated Rodent Heart Experiments
Hearts were obtained from Sprague-Dawley rats (weight, 250 to 500 g), and all animals received humane care in compliance with the ``Guide for the Care and Use of Laboratory Animals'' (NIH publication number 85-23, revised 1985). Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital. Heparin (200 units) was administered intravenously. A median sternotomy was performed, and the hearts were rapidly excised and immersed in chilled normal saline solution. The experimental preparation has been described previously [12]. After excision the hearts were subsequently perfused in a Langendorff apparatus with filtered Krebs-Henseleit buffer at a pressure of 100 cm H₂O. The reservoirs and conduits were placed in a water jacket kept at 37°C. The perfusate was aerated with 95% oxygen and 5% carbon dioxide and the pH adjusted to 7.4.

A saline-filled balloon was inserted into the left ventricle through a left atriotomy and fixed to the mitral valve ring with a pursestring suture. The balloon volume was varied in 0.02-mL increments from 0 to 0.4 mL, but not to exceed an end-diastolic pressure of 30 mm Hg. Data were obtained after a 30-minute stabilization period before storage and after 45 minutes of reperfusion following storage.

The developed pressure was recorded before and after storage at the preischemic balloon volume associated with an end-diastolic pressure of 5 mm Hg. Compliance curves were assessed by linear regression analysis of the end-diastolic pressure data to calculate a slope and X-intercept. Linear regression provided a reasonable model for the diastolic function curves (R² of 0.85 to 0.99 for individual curves). Coronary flow was obtained in duplicate by a timed collection carried out in the emptying beating state. Hearts were rejected for subsequent storage if they exhibited a developed pressure of less than 90 mm Hg (4 animals) or a coronary flow of greater than 30 mL/min (4 animals), or a cardiac arrest occurred during the baseline stabilization period (2 animals).

The adenine nucleotide contents were measured in control hearts after baseline perfusion in the Langendorff apparatus before storage, immediately after storage, or following 45 minutes of reperfusion after storage (6 hearts/group). Hearts were submerged immediately in liquid nitrogen. The purine metabolite levels were measured by high-performance liquid chromatography using a previously described method [13], and results were expressed as micromoles per gram dried weight. The creatine kinase release and lactate dehydrogenase release were assessed for the 45-minute reperfusion period after the storage interval. The entire coronary effluent for the reperfusion phase was collected. Enzyme release was determined spectrophotometrically using a Hitachi Automatic Analyzer 737 and Olympus AU800 at a spectral wavelength of 340 nm. The results of these studies were expressed as international units per gram dried weight.

Control functional data were obtained after 30 minutes of perfusion in the Langendorff apparatus before ischemia. Hearts were preserved with unmodified UWS or UWS supplemented with the optimal NBMPR concentrations identified by the preliminary cell culture studies. After aortic root flushing (15 mL/kg), hearts were stored for 8 hours at 0°C in 15 to 20 mL of storage solution.

Statistical Analysis
Data analysis was facilitated using Statistical Analysis System software (SAS Institute, Cary, NC) and a microcomputer. Variables are expressed as the mean ± standard deviation of the original values, or as a percentage of the control value. Data analysis was performed with a one-way analysis of variance and between-group differences specified with Duncan's multiple-range test. Diastolic function was analyzed additionally using a multivariate analysis of variance, testing simultaneously both the slope and X-intercept. Statistical significance is assumed for a p value of less than 0.05.

	Results

Top Footnotes Abstract Introduction Methods Results Comment Acknowledgments References

 
Human Cardiomyocyte Experiments
The recovery of DNA after storage was reduced for all test solutions (p < 0.0001, analysis of variance) compared with the control results, but was similar among the different groups (UWS, 25.5% ± 9.6%; N1, 27.0% ± 12.1%; N3, 26.3% ± 12.1%; N10, 27.3% ± 10.4%; N30, 27.5% ± 7.3%; N100, 28.5% ± 7.6%) and the NBMPR vehicle group (28.0% ± 12.8%).

Figure 1 depicts the poststorage values of adenine nucleotides. The adenosine triphosphate (ATP) levels were increased in association with NBMPR supplementation, and the improvement was statistically significant for the N1 and N3 groups. The adenosine diphosphate level was unchanged with storage, but the adenosine monophosphate values were increased for all test solutions. The levels of total adenine nucleotides were increased in association with nucleoside-transport inhibition, with an optimal improvement identified for the cells stored in UWS with 3 µmol/L of NBMPR. There was no significant effect noted for the NBMPR vehicle (ATP, 3.87 ± 1.63 nmol/µg DNA; total adenine nucleotides, 5.96 ± 2.74 nmol/µg DNA).

View larger version (42K): [in this window] [in a new window]   Fig 1. . Adenine nucleotide results (mean ± standard deviation; 7 dishes/group) are presented for control samples and after 12-hour storage at 0°C for unmodified University of Wisconsin solution (UWS) or that supplemented with 1, 3, 10, 30, or 100 µmol/L of the nucleoside-transport blocker p-nitrobenzylthioinosine (groups N1, N3, N10, N30, N100). The addition of the blocker augmented the recovery of adenosine triphosphate (ATP) and total adenine nucleotides (TAN), with an optimal improvement observed using 1 to 3 µmol/L. (ADP = adenosine diphosphate; AMP = adenosine monophosphate; N1, N3, N10, N30, N100 = 1, 3, 10, 30, and 100 µmol/L of p-nitrobenzylthioinosine.)

View larger version (41K): [in this window] [in a new window]   Fig 2. . Adenosine, inosine monophosphate (IMP), inosine, and hypoxanthine values were increased after storage with unmodified and modified preparations of University of Wisconsin solution. (See Fig 1 for abbreviations.)

Isolated Rodent Heart Experiments
The developed pressure and coronary flow were similar for all groups before storage (developed pressure: 128.9 ± 16.0 to 133.3 ± 15.4 mm Hg, p = 0.90; coronary flow: 20.3 ± 6.4 to 21.2 ± 3.8 mL/min; p = 0.97). The developed pressure was reduced after storage for all preservation media groups, but increased in the N1 and N3 groups compared with the unmodified UWS group (Fig 3). Coronary flow was decreased after storage for all groups but increased in the N3 group relative to the UWS group (Fig 4). The diastolic function results are presented in Table 1. The poststorage slopes were increased for each of the study conditions but did not differ among the groups. There was no significant change in the X-intercept, although it tended to be decreased for the UWS group and to be increased for the NBMPR groups.

View larger version (37K): [in this window] [in a new window]   Fig 3. . Postreperfusion developed pressure (mean ± standard deviation; 9 to 13 hearts/group) as a percentage of prestorage values. Developed pressure was increased in the nucleoside blocker groups (N1 [group receiving 1 µmol/L of p-benzylthioinosine] and N3 [group receiving 3 µmol/L of p-benzylthioinosine]) compared with group receiving unmodified University of Wisconsin solution (UWS).

View larger version (38K): [in this window] [in a new window]   Fig 4. . Postreperfusion coronary flow results (mean ± standard deviation; 9 to 13 hearts/group) as a percentage of prestorage values. Coronary flow was increased in the group receiving 3 µmol/L of p-nitrobenzylthioinosine in the preservation solution (N3) compared with that receiving the unmodified University of Wisconsin solution (UWS). (N1 = group receiving 1 µmol/L of p-nitrobenzylthioinosine in preservation solution.)

	Comment

Top Footnotes Abstract Introduction Methods Results Comment Acknowledgments References

The primary rationale for the use of cultured human cardiomyocytes stems from the potential concerns about discrepancies introduced by species differences. Second, the use of cultured human cardiomyocytes allows one to perform experiments on specific cell types (eg, myocytes, fibrocytes, and endothelial cells). We have used right ventricular outflow tract myocardium from patients with tetralogy of Fallot as a convenient source of a large amount of ventricular myocardium to initiate the cultures. We emphasize the importance of confirming the cell culture findings in an intact cardiac preparation; however, using similar experimental protocols, we have previously shown that the supplementation of UWS with glucose also improved the poststorage results in the settings of both human cultured cardiomyocytes [14] and isolated rodent hearts [12]. We acknowledge that buffer-perfused systems are not ideal and that blood-perfused models are more physiologic.

There exists considerable interest in the application of UWS to cardiac allograft storage [1–6]. Clinical trials have been conducted in patients in the setting of conventional organ ischemic times, that is, less than 4 hours [15–17]. A reduction in the requirement for electrical defibrillation was noted in all three studies. The trials comparing UWS and Stanford cardioplegia [15, 16] revealed additional evidence for the superiority of the myocardial protection conferred by UWS, as shown by the cardiac levels of ATP or creatine phosphate and by the postoperative enzyme release. The one study that compared UWS and St. Thomas' Hospital solution [17] did not reveal any other evidence of improved preservation with UWS. Studies conducted on organs from high-risk donors by virtue of the prolonged organ ischemic time anticipated, the high level of inotropic agents utilized, small donor size, or advanced age may better discriminate the protective properties of alternative storage solutions.

The beneficial properties of UWS with respect to cardiac storage may relate to the inclusion of adenosine in its formulation [6]. Adenosine originally was documented to facilitate ATP synthesis during hypothermic kidney perfusion [18]. However, the composition of UWS is unique in many regards, and, furthermore, multiple different UWS-like formulations have been employed during experimental evaluations. Recent studies conducted by Lasley and Mentzer [6] directly evaluated the contribution of adenosine to UWS. The resultant interstitial adenosine concentrations were 20 to 40 times greater in the adenosine-treated hearts, as shown by cardiac microdialysis, and functional recovery was enhanced.

Adenosine supplementation has been found to be effective in models of regional ischemia [19], global ischemia [20], cardioplegia [7], ventricular assist [21], and transplantation [6]. Adenosine has been implicated as the mediator of ischemic preconditioning through the process of A₁-selective receptor activation [22]. The ultimate effector of this phenomena is uncertain, although evidence supports the role of pertussis-sensitive G_i proteins linked to K⁺_ATP channels [23]. The potential mechanisms responsible for the improved tolerance to ischemia-reperfusion, extensively covered in previous reviews [24], relate to enhanced tissue adenine nucleotide levels, the modification of glucose metabolism, vascular dilatation, a cardioplegia effect secondary to the depression of nodal and conductive tissue function, an inhibitory activity directed against neutrophils or platelets, or an antiadrenergic effect.

Masuda and associates [9] investigated the role of the experimental nucleoside-transport blocker R75231 for 24-hour cardiac storage. The best results were obtained when R75231 was added to both the cardioplegic-storage solution and the reperfusion medium. Improved recovery was associated with greater levels of ATP and total adenine nucleotides after storage and reperfusion in the nucleoside transport–blocker groups. The adenosine and inosine levels were greater after storage in the control group, but these were not maintained during reperfusion. The hypoxanthine concentrations were increased after storage and increased further during reperfusion in the untreated hearts, but nucleoside-transport inhibition was associated with maintained levels of adenosine and inosine during reperfusion and with minimal hypoxanthine values. Hypoxanthine can act as a substrate for the formation of oxygen free radicals associated with the xanthine–xanthine oxidase reaction.

In our cardiomyocyte experiments, we identified that the addition of 1 and 3 µmol/L of NBMPR to the UWS was associated with increased levels of ATP and total adenine nucleotides after storage. Based on these data, rodent heart experiments were performed with 1 and 3 µmol/L of NBMPR. The adenosine concentrations were markedly elevated in the cellular preparations related to the high adenosine concentrations in the storage solutions, as we have previously reported [3, 22], without any differences identified among the groups. The inosine and hypoxanthine values were similar to those achieved with unmodified UWS.

The NBMPR appeared to be biologically active in the rodent hearts, as the postreperfusion hypoxanthine levels were unmeasurable. The reasons for the discrepancy in the adenine nucleotide levels between the rodent and human cardiomyocyte experiments may relate to model or species differences. A beneficial effect of nucleoside-transport blockade was noted in terms of the developed pressure and coronary flow, as compared with the effects of unmodified UWS. These findings support the addition of NBMPR to UWS and are compatible with the concept that nucleoside-transport blockade is the mechanism responsible for the improved results. The optimal concentration of NBMPR shown by the cell culture experiments was 3 µmol/L. The coronary flow results from the rodent heart experiment recommend the addition of 3 µmol/L of NBMPR. Other end points (tissue levels of creatine phosphate and adenosine, and the cardiac release of creatine kinase and lactate dehydrogenase) suggest that a concentration of 1 µmol/L may be preferable.

In summary, we have provided evidence that the putative nucleoside transport–blocking agent NBMPR enhances the recovery of cardiac tissue stored with UWS. Additional relevant questions that remain unanswered are whether exogenous adenosine is necessary for this benefit and whether supplementing other storage solutions with NBMPR is advantageous.

	Acknowledgments

Top Footnotes Abstract Introduction Methods Results Comment Acknowledgments References

 
Supported in part by the HSFO grant A-1959. Stephen E. Fremes is a Research Scholar of the HSFO.

We thank Dr William G. Williams for assistance with the pediatric myocardial tissue harvesting. We also extend our appreciation to Mara Bajc for assistance in the preparation of the manuscript and Lisa Keaney for the artwork. We are grateful to Peter Meighoo and the staff of the Department of Clinical Biochemistry at the Toronto Hospital for assistance with the cardiac enzyme assays. We also acknowledge the Multiple Organ Retrieval and Exchange Program and Dupont Canada Limited for donation of the University of Wisconsin solution used in these studies.

	Footnotes

Top Footnotes Abstract Introduction Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr Fremes, Sunnybrook Health Science Center, 2075 Bayview Ave-H405, Toronto, Ontario, Canada M4N 3M5.

	References

Top Footnotes Abstract Introduction Methods Results Comment Acknowledgments References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fremes, S. E. Articles by Mickle, D. A. G. Search for Related Content PubMed PubMed Citation Articles by Fremes, S. E. Articles by Mickle, D. A. G.