Conflicts of Interest The authors of this manuscript have no conflicts of interest to disclose.
Moto Fukai MD, PhD, Department of General Surgery, Hokkaido University Graduate School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan. Tel.: +81-11-706-5923; fax: +81-11-706-7064; e-mail: email@example.com
Since prolonged cold preservation of the heart deteriorates the outcome of heart transplantation, a more protective preservation solution is required. We therefore developed a new solution, named Dsol, and examined whether Dsol, in comparison to UW, could better inhibit myocardial injury resulting from prolonged cold preservation. Syngeneic heterotopic heart transplantation in Lewis rats was performed after cold preservation with UW or Dsol for 24 or 36 h. In addition to graft survival, myocardial injury, ATP content, and Ca2+ -dependent proteases activity were assessed in the 24-h preservation group. The cytosolic Ca2+ concentration of H9c2 cardiomyocytes after 24-h cold preservation was assessed. Dsol significantly improved 7-day graft survival after 36-h preservation. After 24-h preservation, Dsol was associated with significantly faster recovery of ATP content and less activation of calpain and caspase-3 after reperfusion. Dsol diminished graft injury significantly, as revealed by the lower levels of infarction, apoptosis, serum LDH and AST release, and graft fibrosis at 7-day. Dsol significantly inhibited Ca2+ overload during cold preservation. Dsol inhibited myocardial injury and improved graft survival by suppressing Ca2+ overload during the preservation and the activation of Ca2+ -dependent proteases. Dsol is therefore considered a better alternative to UW to ameliorate the outcome of heart transplantation.
Graft dysfunction in the early phase of reperfusion, due to ischemia and reperfusion injury (IRI), is still the critical problem to be conquered in clinical heart transplantation . Since the heart is susceptible to cold IRI , the time limit for a safe heart graft is 4–6 h in clinical settings using University of Wisconsin solution (UW) . Improvement of the graft quality after cold preservation is thus a very important issue, but the method of cardiac cold preservation has not been dramatically changed since the UW was introduced in 1988 . For this reason, a better, alternative organ preservation solution is needed.
During cold preservation, harmful processes such as ATP depletion , Ca2+ overload , production of reactive oxygen species (ROS) , cellular acidosis , swelling , and cytoskeletal disruption  are initiated and progress. During subsequent re-warming ischemia and reperfusion, some of these harmful cascades, including ROS production, Ca2+ overload and downstream activation of proteases , and delayed recovery of ATP production , are further enhanced. Prolonged cold preservation exacerbates these processes, and eventually causes cardiac graft injury.
We therefore developed Dsol, a novel organ preservation solution based on UW solution with a high sodium and low potassium component, modified impermeants, and deuterium oxide (D2O) as solvent (Table 1). We expect the extracellular-type composition of this solution without hydroxyethyl starch (HES) to inhibit coronary endothelial injury and subsequent graft infarction after reperfusion [13,14]. In addition, the impermeants sucrose and mannitol, which cost less than raffinose, are expected to give the solution potent cellular protection and antioxidant effects [15,16]. Deuterium oxide (D2O) has unique biological effects, including inhibition of cytosolic Ca2+ overload , and the stabilization of microtubules , actin cytoskeleton , plasma membrane , and membrane-bound proteins . D2O also accelerates ATP production by stimulation of glucose uptake, glycolysis , and mitochondrial respiration . These properties could suppress Ca2+-induced cellular damage, and maintain structural and functional homeostasis of cardiomyocytes. In previous studies, the efficacy of D2O for liver and heart preservation , and D2O-containing solutions for kidney , pancreas , and vascular tissue preservation  has been reported. However, the effects of D2O-containing solution have not yet been explored in a heart preservation and transplantation model.
Table 1. Composition of the preservation solutions.
HES, Hydroxyethyl starch.
Freezing point (°C)
The aims of the present study were to test whether Dsol, in comparison to the widely accepted UW, could better inhibit myocardial injury in extended cold preservation and subsequent syngeneic transplantation of rat hearts.
Materials and methods
Chemicals and reagents
All the chemicals and reagents were of the highest grade commercially available, and purchased from Wako Pure Chemical Co. (Osaka, Japan) unless otherwise noted.
Preparation of preservation solutions
UW solution (Viaspan®) was purchased from Bristol–Myers Squibb Co. (New York, NY, USA). Dsol was developed in our laboratory (Table 1). Deuterium oxide was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). The freezing point of Dsol was 0.3 °C and we confirmed that Dsol would not freeze at 4 °C under the conditions employed herein. All solutions were filtered (0.45 μm) before use.
The experiments were approved by the institutional Animal Care Committee, and were conducted under the guidelines for animal care and use of Hokkaido University. Inbred male Lewis (LEW) rats weighing 250–350 g were purchased from Kyudo Co., Ltd. (Saga, Japan), and were used as both donors and recipients. They were maintained in a specific pathogen-free facility, and were used for the experiments without fasting.
Cell culture and reagents
H9c2 cells (passage 18–25; CRL-1446™; American Type Culture Collection, Rockville, MD, USA) were cultured in DMEM (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) heat inactivated bovine serum (Gibco–Invitrogen, Carlsbad, CA, USA), and penicillin–streptomycin (Gibco), under 95% air/5% CO2 at 37 °C. To assess the cytosolic Ca2+ concentration, a FRET-based Ca2+ indicator, the Premo Cameleon Calcium Sensor™ (Molecular Probes Inc. Eugene, OR, USA), was transduced into the H9c2 cells according to the manufacturer’s instructions. Briefly, the cells were incubated in a growth medium containing an appropriate amount of vector at room temperature for 4 h, then incubated for another 16 h in a fresh growth medium containing expression-enhancer solution. Cells (4 × 104 cells/well) were plated on a 96-well culture plate for fluorescent measurement overnight under the normal growth conditions.
Heterotopic cardiac transplantation
Heterotopic heart transplantation was performed as previously described . Briefly, after anesthetization with isoflurane inhalation, sodium heparin (1000 U/kg) was intravenously administered to the donor. Then, the heart was perfused in situ with 4 °C UW or Dsol from the aorta. The heart was rapidly excised and preserved in each solution at 4 °C. Recipients underwent a mid-line abdominal incision after anesthesia. The ascending aorta and pulmonary artery of the donors were anastomosed to the recipient’s infra-renal abdominal aorta and inferior vena cava, respectively. The warm ischemic time was strictly adjusted to 25 min.
Experimental protocol in vivo
The grafts were transplanted after 24-h cold preservation in UW or Dsol solution (UW24 or Dsol24 group, respectively), 36-h preservation in UW or Dsol solution (UW36 or Dsol36 group, respectively), or no preservation (non-preserved control: NPC group). Graft survival was followed for 7 days. In the 24-h preservation groups, rats were sacrificed at 1 and 24 h after reperfusion (R1h and R24h, respectively). Grafts at the end of 24-h cold preservation (CP24h) in UW and Dsol solution, and normal heart controls (NHC) were also sampled. Graft infarction, apoptosis, serum biochemistry, inflammatory cells infiltration, high energy phosphates content, calpain and caspase 3 activities were assessed. At 7 days after reperfusion, rats were sacrificed to examine the level of graft fibrosis.
Graft survival was examined by palpation through the abdominal wall by two independent examiners in a blinded manner. Graft loss was defined as total stasis or the absence of any wall movement by direct inspection.
Cardiac infarct size was assessed at R1h and R24h by triphenyltetrazolium chloride (TTC) staining as previously described . Briefly, the excised hearts were incubated for 12 min in 1.5% TTC (w/v) in PBS at 37 °C, and fixed in 10% formalin-PBS thereafter. After taking microscopic images, the infarct area was calculated using computerized planimetry.
Graft apoptosis was assessed at R24h by terminal dUTP nick end-labeling (TUNEL) staining as previously described . Nuclei were counterstained with hematoxylin. TUNEL-positive cells were counted in five randomly selected HPFs (magnification ×400) adjacent to the necrotic area, the so-called area at risk, in a blinded manner. Mononuclear cells, cells without myofiber, or cells located at the interstitium were excluded as inflammatory cells. Results were expressed as the average number of TUNEL-positive cells per single HPF.
Infiltration of polymorphonuclear neutrophils (PMNs) and monocytes
The numbers of infiltrating inflammatory cells were assessed by counting the number of PMNs and monocytes at R24h. The grafts were fixed in 10% formalin-PBS, embedded in paraffin, and stained with hematoxylin-eosin (HE) for the PMNs count. Graft samples were also embedded and frozen in an OCT compound. Immunohistochemical (IHC) staining for monocytes/macrophages was performed with a mouse anti-rat CD68 antibody (AbD Serotec, Oxford, UK). Then the samples were incubated with a biotinylated goat anti-mouse IgG secondary antibody (DAKO, Cambridge, UK) and streptavidin-biotin-peroxidase (DAKO) in sequence. Detection of antibody binding was performed with 3,3’-diaminobenzidine (DAKO). Cells were counterstained with hematoxylin.
The numbers of PMNs and monocytes/macrophages were counted in 10 randomly selected HPFs for each section.
High energy phosphates
The levels of tissue adenine nucleotides (ATP, ADP, AMP) before preservation, at the end of 24-h cold preservation, and at R1h were measured as previously described . Grafts were snap-frozen and homogenized in 20 μl/mg of ice cold 0.3 M perchloric acid with 0.01% (w/v) EDTA using a Polytron homogenizer (Kinematica Inc., Bohemia, NY, USA). After centrifugation (2200 g, 10 min, 4 °C), the supernatant was neutralized by 5 N KOH. An aliquot (20 μl) was analyzed by HPLC (Eicom, Kyoto, Japan). The dry-to-wet weight ratio of the tissue was separately measured by lyophilization. Myocardial adenine nucleotides were expressed as micromole per gram dry weight (μmol/g dw). Total adenine nucleotide (TAN) was calculated as the sum of ATP, ADP, and AMP.
Grafts excised at R7d were fixed in 10% formalin-PBS, embedded in paraffin, and stained with Masson’s trichrome. After microscopic images were taken with a BIOREVO BZ9100 fluorescence microscope (KEYENCE, Osaka, Japan), they were processed using computerized planimetry software (KEYENCE). The fibrotic area was expressed as the percentage of the total LV area.
Calpain and caspase 3 activation
To determine the levels of activation of calpain and caspase 3, calpain-specific cleavage of cytoskeleton-bound proteins (α-fodrin and talin) and cleavage of caspase 3 were assessed by a standard Western blot analysis [31,32]. The graft was homogenized with a glass-Teflon homogenizer in 4 ml/g of lysis buffer containing 25 mmol/l Tris–HCl, 150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/l EDTA, and 1% protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA) at pH 7.6. The homogenate was centrifuged for 15 min at 14000× g and 4 °C. The protein concentration of the resulting supernatant was determined with a bicinchoninic acid assay (Thermo Scientific, Rockford, IL, USA). Then, the proteins were separated with standard SDS–PAGE techniques. After transfer to a PVDF membrane, the proteins were probed with mouse anti-α-fodrin mAb (1:1000; Biomol, Plymouth Meeting, PA, USA), mouse anti-talin mAb (1:200; Sigma), and rabbit anti-caspase 3 Ab (1:1000; Cell Signaling, Danvers, MA, USA). Then, IgG-horseradish peroxide-conjugated anti-mouse or anti-rabbit secondary antibody (1:2500–1:10000; Amersham Bioscience, Buckinghamshire, UK) was applied for chemiluminescence detection (Amersham Bioscience). α-tubulin was detected with rabbit mAb to α-tubulin (1:1000; Cell Signaling) as an internal control. The cleaved bands of α-fodrin and talin were then normalized by the respective intact bands. Cleaved bands of caspase 3 were normalized by α-tubulin. The values were finally expressed as a percentage of the value in the normal heart controls.
Cytosolic Ca2+ concentration in vitro
Cells expressing a FRET based Ca2+ indicator, Premo Cameleon Calcium Sensor™, were subjected to 24-h cold preservation in UW or Dsol. Cameleon was excited at 370 nm to produce fluorescence from CFP detected at 480 nm in the Ca2+ -unbound form. In the Ca2+ -bound form, FRET occurred from CFP to YFP, resulting in the production of additional fluorescence at 535 nm. The mean fluorescent intensity at 535 nm (MFI535) was expressed as a percentage of the MFI535 before preservation.
Data were expressed as the mean ± standard deviation or mean ± standard error of the mean as annotated. Graft survival was plotted by the Kaplan–Meier method, and was applied to a log-rank test for comparisons. One-factor anova followed by post hoc test was applied as appropriate. A value of P < 0.05 was considered statistically significant.
Dsol ameliorated graft survival
All hearts in the non-preservation control group (NPC) survived for 7 days (Fig. 1). In the 24-h cold preservation experiment, the rate of 7-day graft survival in the Dsol group was 100% (5/5), versus 80% (four of five) in the UW group. In the 36-h preservation experiment, the rate of 7-day graft survival was 75% (six of eight) in the Dsol group, whereas it was only 14% (one of seven) in the UW group (P <0.05; Dsol36 vs. UW36).
Dsol decreased graft infarction, apoptosis, LDH and AST release
Graft infarction at 1 h after reperfusion (R1h) was not evident in all groups, and ranged from 0% to 3.4% of the total LV area. At R24h, the infarct area was 67.8% ± 16.5% of the total LV area in the UW group, whereas it was 11.7% ± 7.3% in the Dsol group (P <0.05; Dsol vs. UW; Fig. 2a).
TUNEL-positive cells, i.e. apoptotic cardiomyocytes, were not found in the NPC group at R24h. The number of TUNEL-positive cardiomyocytes at R24h was significantly smaller in the Dsol group (5.97 ± 2.44 counts/HPF) than in the UW group (15.1 ± 1.30 counts/HPF, Fig. 2b).
Serum LDH and AST levels in the UW group (1282 ± 667 and 1144 ± 427 IU/l, respectively) were significantly higher than those in the Dsol group (516 ± 195 and 463 ± 120 IU/l, respectively) at R24h (Fig. 2c).
Dsol reduced graft fibrosis
The fibrotic area at R7d was significantly larger in the UW group (39.5% ± 11.0%) than in the Dsol group (20.7% ± 11.1%) or NPC group (6.9% ± 1.8%, P < 0.05 for UW versus NPC and for UW versus Dsol, Fig. 2d).
Dsol suppressed the infiltration of inflammatory cells
The number of polymorphonuclear neutrophils (PMNs) in the interstitium at R24h was significantly higher in the UW group (12.4 ± 1.37 counts/HPF) than in the Dsol group (4.5 ± 0.24 counts/HPF). The number of CD68-positive monocytes/macrophages at R24h was significantly higher in the UW group (21.7 ± 1.76 counts/HPF) than in the Dsol group (9.6 ± 0.87 counts/HPF, Fig. 2e).
Dsol improved the restoration of high energy phosphates after reperfusion
ATP content in the normal heart was 7.87 ± 1.86 (μmol/g dw), whereas ATP was not detected at the end of the 24-h cold preservation in either group. At R1h, it was significantly higher in the Dsol group (8.34 ± 2.16 μmol/g dw) than in the UW group (4.32 ± 2.90 μmol/g dw, Fig. 3). TAN was also significantly higher in the Dsol group (15.94 ± 1.89 μmol/g dw) than in the UW group (11.77 ± 3.39 μmol/g dw).
Dsol inhibited cold preservation-induced Ca2+ overload in vitro
After 24-h cold preservation, MFI535 increased to as much as 376% of the basal level in the UW group, whereas it increased to only 140% of the basal level in the Dsol group (P < 0.0001). Therefore, Dsol inhibited Ca2+ overload during cold preservation (Fig. 4a).
Dsol inhibited calpain and caspase-3 activation
The calpain-specific substrates, talin and α-fodrin, were not cleaved at the end of the 24-h cold preservation period in either the UW or Dsol group (Fig. 4b). At R1h, they showed a significantly greater amount of cleavage in the UW group compared to the normal heart control group (NHC). Calpain-mediated cleavage was significantly suppressed in the Dsol group (P < 0.05 vs. UW, Fig. 4b).
The activations of caspase 3 by cleavage were assessed. The active cleaved fragments of caspase 3 (17 kDa) were significantly increased at R1h in the UW group compared to the NHC group (P < 0.05, vs. NHC), whereas they were significantly suppressed in the Dsol group (P < 0.05, vs. UW, Fig. 4c).
In the current study, we demonstrated that the novel organ preservation solution Dsol improved cardiac graft survival after 36-h cold preservation. After 24-h preservation, Dsol markedly suppressed necrosis and apoptosis as compared to UW solution. Dsol also enabled rapid restoration of the high energy phosphate that had been exhausted from the grafts during the preservation period. Dsol was clearly shown to prevent the elevation of cytosolic Ca2+ concentration during cold preservation in vitro, and inhibited Ca2+ -dependent activation of calpain and subsequent activation of caspases-3, compared to UW solution in vivo. These data clearly demonstrated the advantage of Dsol over UW, with the former showing excellent inhibition of cardiac graft injury after prolonged simple cold static preservation and subsequent cardiac transplantation in rats.
In previous reports using the same model, the graft function of the UW-preserved rat hearts after transplantation was recovered in the 12-h preservation group , whereas it was impaired in the 18-h preservation group . Further, 24-h preservation in UW raised the possibility of graft loss due to the critical ischemia/reperfusion injury . Infarction of grafts after prolonged cold preservation presents a risk of graft loss. To avoid graft loss in such cases, previous reports have suggested the importance of suppressing graft infarction to below 15% of the total area of individual grafts after reperfusion [35,36]. In the present study, Dsol suppressed the graft infarction in just 11% of total area of grafts, and prevented graft loss completely. On the other hand, UW induced 68% graft infarction and resulted in graft loss in 20% of grafts after 24-h cold preservation. In addition, the surviving grafts in the UW-preserved group tended to beat more weakly than the Dsol-preserved grafts. However, we could not evaluate graft function in this study because we employed a non-functional model. Functional assessment using a functional model remains a challenge for future studies. However, the present results do indicate that Dsol has a more powerful protective effect than UW solution, although this protective effect appeared more evident after 36-h preservation.
Necrosis at the center of the infarction and apoptosis around the necrotic area, the so-called area at risk (AAR), are closely related to graft survival and contractile function . After prolonged cold preservation and reperfusion, cardiomyocytes fell into necrosis for various reasons, including hypercontracture, insufficient blood flow due to vascular failure, and activation of necrosis-inducing proteases [14,38,39]. In the present study, UW could not prevent necrotic cell death, as demonstrated by TTC staining, AST and LDH release, and eventual graft fibrosis, which was consistent with a previous report , whereas Dsol achieved nearly complete inhibition. Necrotic cardiomyocytes induced infiltration of inflammatory cells in the UW group but not in the Dsol group. These cells, in turn, damage viable cardiomyocytes by secreting inflammatory mediators . Therefore, the prevention of necrosis also has important implications in terms of stopping this harmful cycle. Cardiomyocytes that manage to just avoid necrosis often fall into apoptotic cell death within the AAR . We demonstrated that abundant TUNEL-positive apoptotic myocardia were found at the AAR in UW-preserved hearts, whereas they were significantly suppressed in the Dsol group. Dsol prevented cell death not only by preventing necrosis but also by preventing apoptosis.
Cytosolic Ca2+ overload during prolonged cold preservation and Ca2+ -dependent activation of calpain and caspases after reperfusion play a central role in cellular necrosis and/or apoptosis. Calpain is activated by cytosolic Ca2+ overload, and activated calpain, in turn, induces necrosis by cleavage of cytoskeletal proteins such as α-fodrin and talin . Calpain also triggers apoptosis by caspase-12 activation , and Bid  and Bax cleavage , followed by caspase 3 activation. Among the many unique properties of D2O, such as stabilization of the microtubules , actin cytoskeleton , plasma membrane , and membrane-bound proteins , we focused on the ability of D2O to suppress the elevation of cytosolic Ca2+ concentration . D2O is reported to inhibit calcium influx via the plasma membrane L-type Ca2+ channel  as well as calcium efflux from the sarcoplasmic reticulum (SR) to the cytosol . Our present in vitro study demonstrated that cytosolic Ca2+ concentration was elevated up to 3.8-fold after 24-h cold preservation in the UW group. Elevated cytosolic Ca2+ at the end of the cold preservation period in turn leads to the activation of Ca2+ -dependent proteases, and thereby protease-induced necrosis and apoptosis of cardiomyocytes after reperfusion. In this study, the major source of augmented cytosolic Ca2+ during preservation should be the efflux from SR, because both UW and Dsol are Ca2+ -free solutions. The D2O present in the Dsol could inhibit Ca2+ release from SR and suppressed the elevation of cytosolic Ca2+ concentration during cold preservation. Accordingly, Dsol dramatically suppressed the activation of these degradative Ca2+ -dependent proteases thereafter. This property of D2O should be a key mechanism of the graft protection with Dsol.
In addition to cellular death, the energy state, which is established mainly by mitochondrial oxidative ATP production, is closely related to the cardiac kinetics after transplantation. Flameng et al. reported that the impairment of ATP restoration after reperfusion, even if the ATP content was maintained at the end of 24-h cold static preservation, causes cardiac contractile dysfunction after transplantation . Although Dsol failed to preserve ATP content during cold preservation in the present study, rapid recovery of ATP content was clearly shown at 1 h after reperfusion. Meanwhile, UW failed to recover ATP synthesis, even though graft infarction was not evident.
Although the intracellular-type component and HES adopted by UW can potently prevent cellular swelling during cold preservation, they tend to induce graft infarction as a result of coronary endothelial injury [13,14]. Therefore, we adopted the extracellular-type component without HES for Dsol. In this respect, the concept of Dsol is similar to that of Celsior , which showed better preservation than UW within a relatively short period , but not after extended cold preservation [48,49]. The reasons for the potent protection by Dsol even after a prolonged period could be the modified impermeants and D2O, which could compensate for the demerits of the extracellular-type composition. Modified impermeants such as mannitol and sucrose, which per se have cyto-protective  and anti-oxidative effects , could reduce organ swelling. Other properties of D2O, in addition to the inhibition of Ca2+ -overload, such as stabilization of the microtubules , actin cytoskeleton , plasma membrane , and membrane-bound proteins , could help Dsol to inhibit graft injury.
In conclusion, Ca2+ overload initiated during cold preservation induces the activation of harmful proteases, and subsequent apoptosis and necrosis of cardiomyocytes after reperfusion, finally leading to graft loss. A novel organ preservation solution, Dsol, was shown to be superior to UW solution at inhibiting myocardial injury during extended cold preservation and subsequent syngeneic transplantation of rat hearts by inhibiting Ca2+ overload during cold preservation and subsequent activation of proteases. This solution could reduce the mortality of heart transplantation. Moreover, the protective effect of this solution could prolong the safe preservation time of cardiac grafts and increase the opportunities for organ distribution.
KW, MF, KY and ST: designed the experiments. KW and MF: wrote the article. KW, MF, TK, GH, SS and DF: contributed to the acquisition of data and analysis. SH, TS, MT, TS and HF: provided expertise. MF and MS: provided new reagents. KW, MF, KY, TK and ST: interpreted the data.
This study was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (No. 2224904800), and by grants-in-aid for Young Scientists (2006-103) and for Scientific Research (2007-87, 2008-33) from the Regional R&D Proposal-Based Program of the Northern Advancement Center for Science & Technology of Hokkaido, Japan.
We thank Keyence Co. (Osaka, Japan) for their excellent technical advice and support in obtaining the pathological images.