Ann Thorac Surg 2001;71:1290-1295
© 2001 The Society of Thoracic Surgeons
Original article: cardiovascular
Cell volume and ionic transport systems after cold preservation of coronary endothelial cells
Juliana Redondo, PhDa,
María E. Pacheco, BSca,
Ana M. Manso, BSca,
Mercedes Salaices, PhDa,
Jesús Marín, MD, PhD*,a,b
a Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain
b Servicio de Farmacología Clínica, Hospital Clínica Puerta de Hierro, Madrid, Spain
Accepted for publication November 19, 2000.
Address reprint requests to Dr Redondo, Departamento Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain
e-mail: julia.redondo{at}uam.es
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Abstract
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Background. Hypothermia-induced changes in cell volume and ionic transport systems of coronary endothelial cells may play a role in the development of coronary artery disease in cardiac transplant recipients.
Methods. Coronary endothelial cells were incubated in University of Wisconsin solution or culture control medium for up to 48 hours at 4°C. Parallel control cultures were incubated at 37°C. Na/K-ATPase and Na/K/Cl cotransport activities were determined as ouabain- and furosemide-sensitive 86Rb+ uptake, respectively. Cell volume changes and cell death were analyzed by a FACScan flow cytometer and the release of lactate dehydrogenase, respectively.
Results. Coronary endothelial cells stored in University of Wisconsin solution up to 6 hours showed an increased Na/K-ATPase activity compared to control cells, whereas no changes were observed in Na/K/Cl cotransport activity or cell volume. Long-term preservation (24 and 48 hours) was associated with a partial loss of cell viability, as demonstrated by lactate dehydrogenase release, and dramatic alterations in ionic transport system activities.
Conclusions. University of Wisconsin solution seems to prevent coronary endothelial cells Na/K/Cl cotransport activity changes during cold preservation, which could alter cell volume regulation and cause cell injury.
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Introduction
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Hearts are stored at 4°C to minimize hypoxic injury to the grafts during the time needed for transport and for recipient preparation [1]. However, although hypoxic injury is greatly diminished by cold storage [2], hypothermia itself can lead to cellular injury [3, 4]. The cellular compartment most sensitive to preservation damage seems to be the vascular endothelium [3, 5]. The endothelial cell of the coronary vasculature is vital in the regulation of blood vessel wall structure, vasomotor tone, and thrombogenicity [3]; therefore, alterations in both the physiologic and biochemical dynamics of endothelial cells may lead to unsuccessful heart transplantations. In fact, early endothelial dysfunction has been correlated with the development of transplant coronary artery disease at 1-year after transplant [6].
Exposure of endothelial cells to cold temperatures produces a loss of transmembane ion gradients and membrane barrier functions as well as changes in cell volume [7]. Regulation of endothelial cell volume is mainly controlled by the Na/K/Cl cotransport system and the Na/K-ATPase [8]. It has been reported that low temperatures cause the inhibition of the Na/K-ATPase, leading to the intracellular accumulation of sodium and subsequently of chloride, a process that is followed by cell swelling, considered to be the main factor contributing to cold-induced cell injury [7, 9]. In the case of the Na/K/Cl cotransport system there is also wide experimental evidence showing its importance in the control of cell volume regulation [8, 10]; however, little is known about how cold storage of cells may affect this ionic transport.
Taking into account the changes in cellular homeostasis and in cell volume during cold storage, different preservation solutions have been designed to minimize these alterations [1]. The University of Wisconsin (UW) [11–13] and the Brettschneider’s histidine-tryptophan-ketoglutarate [14–16] solutions are commonly used for cardiac preservation. However, the UW solution can certainly be considered the current golden standard solution, as it is the most used preservation fluid for different organs with excellent clinical and experimental preservation data [17]. Nevertheless, even with UW solution, cold-induced cell volume changes and imbalances in cellular ion homeostasis seem to occur [9, 18]. Moreover, cold preservation time with this fluid remains an important determinant to keep the cell integrity and the occurrence of delayed graft function [18].
In the present work, we have studied the effects of cold storage in UW solution of porcine coronary endothelial cells (CEC) on the Na/K-ATPase and the Na/K/Cl activities, as well as the possible changes in endothelial cell volume and viability after cold preservation in UW solution.
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Material and methods
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Cell isolation
Endothelial cells were obtained from left anterior descending coronary artery. Porcine hearts were obtained immediately after slaughter and placed in ice-cold phosphate-buffered saline (Biological Industries, Beit Hamek, Israel). The arteries were removed and cleaned of extraneous muscle and connective tissue using sterile techniques, cut open longitudinally to expose the lumen, and pinned with the lumen side up on silicone elastomer in a 35-mm tissue culture dish in a vertical laminar air flow cabinet. The tissue culture dish contained Dulbecco’s modified Eagle’s medium (Biological Industries) containing 0.1% bovine serum albumin (Sigma Chemical Co, St. Louis, MO) and 2 mg/mL collagenase (type II, Worthington Biochemical, Lakewood, NJ). The dish was incubated for 30 minutes at room temperature in the flow cabinet. After centrifugation at 800 revolutions/min for 3 minutes, the supernatant was removed, and the pellet was resuspended in tissue culture medium supplemented with 20% fetal calf serum (Biological Industries), 100 U/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin B, and 0.5 µg/mL endothelial growth factor. The medium containing the cells was added to 25-cm2 culture flasks coated with 1% gelatin. Cells were characterized as endothelial cells by immunocytochemical staining with a monoclonal antibody QBEnd/10 raised against a CD34 endothelial marker. Three or more different CEC cultures were used for each type of experiment; every one of these cultures being obtained from porcine coronary arteries of four different animals. Coronary endothelial cells were cultured by light trypsinization and identically treated cell lines between passages 3 and 10 were used for experiments.
Preservation
Coronary endothelial cells were cultured until they reached confluence and then were growth arrested by replacement of culture medium with serum-free Dulbecco’s modified Eagle’s medium containing 0.1% bovine serum albumin, antibiotics, and 15 mmol/L HEPES (control medium) for 24 hours. The medium with the dead cells was then removed by aspiration. Afterward, fresh control medium (electrolyte composition: 2 mmol/L CaCl2, 0.4 µmol/L Fe(NO3)3, 5.4 mmol/L KCl, 0.8 mmol/L MgSO4, 110 mmol/L NaCl, 44 mmol/L NaHCO3, 0.9 mmol/L NaH2PO4 at pH 7.4) or unmodified UW solution (29 mmol/L Na+, 125 mmol/L K+, 5 mmol/L Mg2+, 25 mmol/L H2PO4-, 5 mmol/L SO42-, 100 mmol/L lactobionate, 30 mmol/L raffinose, 50 g/L hydroxyethyl-starch, 1 mmol/L allopurinol, 5 mmol/L adenosine, 3 mmol/L glutathione at pH 7.4; before use, the solution was supplemented with 40 U/L insulin, 18 mg/L dexamethasone and 2 x105 U/L penicillin) were added to the plates. Cells were stored at 4°C for different time periods in a refrigerator under room air conditions. Parallel cultures were stored in control medium at 37°C in the incubator and used as a control.
Na/K-ATPase and Na/K/Cl cotransport activities
Coronary endothelial cells were plated into 24-well plates and treated as previously described. Na/K-ATPase and Na/K/Cl cotransport activities were determined by using 86Rb+, a radioactive analogue of K+, as previously described [19, 20]. Na/K-ATPase activity was estimated as the difference between 86Rb+ uptake in the absence and the presence of ouabain (ouabain-sensitive 86Rb+ uptake), a Na/K-ATPase inhibitor. In a similar way, Na/K/Cl cotransport activity was determined as furosemide-sensitive 86Rb+ uptake by using the cotransporter blocker furosemide. After different time preservation periods, the medium was removed and replaced by control medium either alone or containing 1 mmol/L ouabain or 100 µmol/L furosemide. The cells were preincubated at 37°C for 15 minutes; then 3.8 µmol/L 86Rb+ (1 µCi/ml; specific activity, 4.68 mCi/mg; New England Nuclear, Boston, MA) was added to every well, and the cells were incubated at 37°C for 40 minutes, which has been previously determined as an optimal period to value the Na/K-ATPase and Na/K/Cl cotransport activities [19]. Afterward, the medium was aspirated, and cell layers were rapidly rinsed six times with 100 mmol/L ice-cold MgCl2. Intracellular 86Rb+ was extracted with 500 µL of 5% trichloroacetic acid at 4°C for 1 hour. Aliquots of 400 µL from every well were used for determination of 86Rb+ by counting in a Beckman LS2800 liquid scintillation counter (Beckman Instruments, Fullerton, CA). After removal of trichloroacetic acid, cell protein was solubilized by incubation with 200 mmol/L NaOH at 4°C overnight. Protein determination was performed according to the method described by Bradford with use of bovine serum albumin as standard. 86Rb+ uptake was expressed as nanomoles per milligram of protein.
Lactate dehydrogenase activity
Cells were seeded in 24-well plates at a density of 50,000 cells/well. Three days later, the cells were growth arrested by replacement of culture medium by control medium for 24 hours. The medium was then removed, and the cells were preserved for different time periods as described above. Afterward, cell-free supernatants were recovered and lactate dehydrogenase (LDH) activity was measured by spectrophotometry, using NADH and sodium pyruvate as substrates. Cell protein determination per well was performed according to the method described by Bradford and LDH activity was expressed as nanomoles per minute and microgram of protein.
Cell volume determination
Cells were seeded in six-well plates and grown until confluence. After a 24-hour growth arrest period, cells were preserved in the different media for 6 or 24 hours. Cell cultures were then gently trypsinized, fixed with 80% ethanol, and stored at 4°C until used for the experiments. Before use, cells were centrifuged for 10 minutes at 1,000 rpm and resuspended in phosphate-buffered saline at 105 cells/mL. Forward scattering and side angle scattering were determined in ethanol-fixed cells by using a FACScan (Becton-Dickinson, Mountain View, CA) flow cytometer equipped with an air-cooled argon laser (15 mW, 488 nm) and a 530-nm band pass for fluorescence. At least 10,000 cells were recorded for each sample. Mean channel numbers were determined on a linear scale throughout these experiments. Cell volume results are expressed in arbitrary units.
Drugs and statistical evaluation
Ouabain octahydrate, furosemide, amphotericin B, endothelial growth factor, NADH, sodium pyruvate, and all other chemicals were obtained from Sigma Chemical, except when stated otherwise.
Data are given as means ± standard error of the mean of the different cultures used for the experiments. Treatments were assayed in triplicates (replicates) for each CEC culture and their mean was considered as a single observation for the statistical analysis. Mean values from replicate wells of the different cultures used were then averaged to be represented in the figures. Data obtained from two groups were compared by means of Student’s t test for unpaired observations and comparisons among multiple groups were performed using two-way analysis of variance for repeated measures. A probability value less than 0.05 was considered significant.
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Results
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Effect of cold storage on LDH release
The release of LDH into the suspension medium was used a measure of cell death of CEC. A time-dependent increase on the amount of LDH released was observed in cells incubated at 4°C and 37°C, independently of the used medium, as reflected in Table 1. However, the loss in cell viability was greater when the cells were stored at 4°C compared to those incubated at 37°C (Table 1). Coronary endothelial cells were better preserved in UW solution than in control medium at 4°C since 2-hour cold storage produced a more pronounced cell death in cultures incubated in control medium than in UW solution (Table 1). Maximum loss of viability was observed in cells treated with control medium at 4°C for 48 hours (Table 1).
Effect of cold storage on Na/K-ATPase and Na/K/Cl cotransport activities
The main ionic transport systems for the control of endothelial cell volume are the Na/K-ATPase and the Na/K/Cl cotransporter [10]. For that reason, we decided to measure the activities of both transporters for 40 minutes after cold storage of CEC in UW solution and control medium. Control cultures stored at 37°C showed no significant change on Na/K-ATPase activity, measured as ouabain-sensitive 86Rb+ uptake, after all studied incubation periods, as shown in Figure 1. However, cold storage of CEC induced a time-dependent increase on Na/K-ATPase activity up to 8-hour incubation in UW solution (maximum response, 385 ± 13.2 nmol 86Rb+/mg protein) and up to 24-hour incubation in control medium (maximum response, 686 ± 22.3 nmol 86Rb+/mg protein) (Fig 1). After 48-hour cold storage in any of both media, a reduced Na/K-ATPase activity was observed (Fig 1), likely due to an important loss of cell viability as suggested by LDH release results (Table 1).
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Fig 1. Effect of preservation of porcine coronary endothelial cells in culture control medium (CM) or University of Wisconsin (UW) solution at 4°C for different time periods on the Na/K-ATPase activity, measured as the ouabain-sensitive 86Rb+ uptake for 40 minutes, after the cold storage. Parallel cultures were incubated in control medium at 37°C. Data are means ± standard error of the mean of four different coronary endothelial cell cultures and each treatment was analyzed in triplicate. *p < 0.05 versus control medium at 37°C;  p < 0.05 compared to control medium at 37°C, and control medium at 4°C.
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Cells incubated in control medium at 37°C showed similar Na/K/Cl cotransport activities, measured as furosemide-sensitive 86Rb+ uptake, after the different time periods of incubation, as shown in Figure 2. In a similar way, no changes were observed in this cotransport activity when the CEC were stored in UW solution at 4°C, although a reduced cotransport activity was observed, after all periods of incubation, compared to cells maintained in control medium at 37°C (Fig 2). On the contrary, CEC treated with control medium at 4°C showed a time-dependent increase on furosemide-sensitive 86Rb+ uptake up to 6 hours of storage (Fig 2). Longer treatment of cells at 4°C (24 and 48 hours) produced a marked decrease on the cotransport activity until reaching similar values to those found in cells incubated at 37°C after 48 hours of incubation (Fig 2).
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Fig 2. Effect of preservation of porcine coronary endothelial cells in culture control medium (CM) or University of Wisconsin (UW) solution at 4°C for different time periods on the Na/KCl cotransport activity, measured as the furosemide-sensitive 86Rb+ uptake for 40 minutes, after the cold storage. Parallel cultures were incubated in control medium at 37°C. Data are means ± standard error of the mean of four different coronary endothelial cell cultures and each treatment was analyzed in triplicates. *p < 0.05 compared to control medium at 37°C, and control medium at 4°C; p < 0.05 compared to control medium at 37°C, and University of Wisconsin solution at at 4°C.
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Effect of cold storage on cell volume
Cells treated for 6 or 24 hours in control medium at 37°C or UW solution at 4°C showed no changes on cell volume (Fig 3). However, CEC stored in UW solution presented a smaller volume than control cells incubated at 37°C (Fig 3). When the cells were kept at 4°C in control medium for 6 hours, an increase on their cell volume was observed, although longer incubation (24 hours) in the same medium produced a reduction on cell size up to similar values than those found in control cultures at 37°C (Fig 3).
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Fig 3. Effect of preservation of porcine coronary endothelial cells in culture control medium (CM) or University of Wisconsin (UW) solution at 4°C on cell volume, measured in arbitrary units, after 6 and 24 hours of cold storage. Parallel control cultures were stored in control medium at 37°C. After incubation, cells were fixed with 80% ethanol and processed for a FACScan flow cytometer. Data are means ± standard error of the mean of four independent experiments. *p < 0.05 compared to control medium at 37°C, and control medium at 4°C; p < 0.05 compared to control medium at 37°C, and University of Wisconsin solution at 4°C; xp < 0.05 versus 6 hours of cold storage.
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Comment
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There is an increased recognition of the importance of an effective CEC preservation in cardiac transplantation. However, little is known about the effects of hypothermia on CEC ionic homeostasis and volume changes [1, 9].
Studies in cardiac transplantation have demonstrated that UW solution is a superior agent in heart preservation compared to other extracellular preservation solutions [11, 12, 21], although the reasons for the different results obtained with the UW solution are not entirely clear. Impermeant agents in the composition of this solution, such as raffinose and lactobionic acid, and the colloid hydroxyethyl starch (pentarstarch), seem to be important in suppression of hypothermia-induced injury to endothelial lining cells, in particular cases of cell swelling [1]. However, despite of these components, changes in cell ionic homeostasis and volume seem to occur after cold preservation in UW solution [9].
Low temperatures appear to cause alterations of cellular ion homeostasis, primarily through inhibition of the Na/K-ATPase [7, 9, 18] leading to an increase in intracellular sodium and subsequently water, a process that is followed by cell swelling. In fact, some researchers have found an increased passive sodium influx in erythrocytes [22] and umbilical vein endothelial cells [18] during cold preservation in culture medium. In the present study, CEC stored in either cold control medium or UW solution up to 6 hours showed an increased Na/K-ATPase activity when such activity was measured after the cold preservation period. These results agree with the above hypothesis that proposes an augmentation on sodium influx during cold preservation [7, 18, 23], which, subsequently, could lead to an activation of the enzyme. Other investigators have found that incubation of cultured liver endothelial cells in cold UW solution or culture medium produced a marked decrease in the cytosolic sodium concentration [9], which would contradict the present findings, as a subsequent inhibition of the enzyme would be expected instead of an activation. However, such decrease in the sodium content seems to be cell-type specific [9, 23], and therefore our results suggest more likely that an increase on intracellular sodium occurs in cultured CEC during cold preservation. Moreover, because similar changes on Na/K-ATPase activity were observed in cells stored in either cold control medium or UW solution, our results suggest that such effect does not appear to be specific of the composition of the UW solution but of the hypothermia itself.
Long-term storage (24 and 48 hours) of CEC at 4°C induced dramatic changes on Na/K-ATPase activity, this effect being likely related to a great cell damage. Thus, the increased Na/K-ATPase activity of CEC stored in cold control medium could be explained by a massive Na+ influx during the cold preservation period, which has been related to the development of cell necrosis [24] and long reperfusion recovery of the transplanted heart [25]. Cells preserved with UW solution showed a gradual decrease on Na/K-ATPase activity, suggesting that components of this solution might be acting to reduce sodium influx, and therefore maintaining cell viability. In accordance to this, an important loss of cell viability, measured as LDH release, was observed in cells incubated with cold control medium compared to those stored in UW solution. However, although the UW solution seems to be adequate for long preservation of abdominal organs [1], it has not been equally used for extended cold storage of the heart. As mentioned above, CEC are crucial for the success of cardiac transplantation, and therefore further studies are required to determine the effects of the unique components of this solution on these cells after long preservation periods.
In addition to the Na/K-ATPase, another main ionic transport system in the maintenance of endothelial cell ionic homeostasis, and therefore cell viability, is the Na/K/Cl cotransport [10]. This transport system plays a major role in vascular endothelial cell volume regulation [10], which is crucial for cell survival and the maintenance of the endothelial barrier function [26]. However, no experimental data on how cold preservation affects this ionic transport system of CEC have been published, although some studies have shown the importance of the Na/K/Cl cotransport in organ preservation [27–29]. Addition of furosemide, an inhibitor of Na/K/Cl cotransport, to the preservation solutions seems to improve long-term organ preservation [27–29], likely by attenuating cell swelling [27–29]. A reduced activity of this cotransporter has been correlated to cell shrinkage, whereas its activation is usually accompanied by cell swelling [10, 30]. In accordance to this, in the present study, the augmented CEC Na/K/Cl cotransport activity after 6 hours of cold storage in control medium was accompanied by cell swelling, likely due to a gradual increment on sodium and water entry during the hypothermic period. Coronary endothelial cells preserved in UW solution showed a reduced cell volume and a diminished Na/K/Cl activity compared to control cells, suggesting that components of this solution might be inducing certain cell shrinkage instead of cell swelling. Although an increase in sodium influx into CEC stored in UW solution seems to occur during hypothermia, subsequent water entry and increase on cell volume appear to be prevented by the components of the UW solution.
Long hypothermic storage of CEC in UW solution induced no significant changes on either cell Na/K/Cl activity or volume, whereas cold control medium produced a marked reduction in this cotransport activity and CEC volume. These alterations in CEC volume could result in significant changes in endothelial barrier permeability to water and solutes by altering the dimensions or structure of the intercellular spaces [10] and cellular organelles [3], leading to a loss in cell viability. In agreement with this, long-term cold storage of our cultures in control medium induced a marked decrease on cell survival, which might be related to volume alterations observed in these cells.
In summary, our data show that cold storage of CEC in either cold control medium or UW solution up to 6 hours produces an increase on Na/K-ATPase activity, likely due to sodium influx during the hypothermic period. This increment on intracellular sodium is probably followed by water entry and a subsequent cell volume increase in CEC stored in cold control medium. However, in cells preserved in UW solution, no alterations on cell volume and Na/K/Cl activity were observed, suggesting that components of this solution are able to avoid subsequent sodium influx-induced cell volume changes. Nevertheless, because UW solution fails to prevent sodium entry into CEC, some modifications in its composition may be needed to avoid other potential alterations associated with hypothermia-induced sodium influx.
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Acknowledgments
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We gratefully thank Carmela Cales, PhD, for her kind collaboration in the flow cytometer experiments and Amelia Torralba, MD, for providing the UW solution. This work was supported by grants from FIS (98/0074–02), D.G.I.C.Y.T. (PM97–0008), Comunidad Autónoma de Madrid (08–3/0003/1998) and Bayer España.
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Footnotes
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* Deceased. 
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Abstract
Introduction
