Heart transplantation is considered the optimal therapy for the treatment of end-stage heart failure or severe coronary artery disease. However, owing to a critical shortage of donor organs, approximately 20 people die each day waiting for life-saving organ donation in the United States.1 In the meantime, a great number of potential donor hearts are discarded because the short safe preservation time of 4–6 hours is exceeded (4°C cold ischemic time).2 In addition, the current preservation strategies potentially compromise organ quality because of ischemia–reperfusion injury, which is associated with considerably high rates of postoperative complications, such as a delayed graft function, rejection, and decreased graft survival.3,4 The development of new preservation methods is desired to increase the availability and quality of organs.
At present, static cold storage of hearts by simple immersion in low-temperature (4°C) crystalloid preservation solutions following cardioplegia is the gold standard preservation method. University of Wisconsin (UW) solution is reported to be the optimal and most commonly used heart preservation solution.5-7 Hypothermia inhibits the metabolic activity and demand of donor organs during preservation. Theoretically, lowering the donor organ temperature by 10°C would reduce the metabolic demand 1.5- to 2.0-fold, and at 4°C, the metabolic demand of the organ would decrease to approximately 5%–10% of the normal demand.8 This standard hypothermic preservation method is simple and cheap but suboptimal for preserving cardiac grafts, especially in the case of marginal donor hearts, which are increasingly used. Researchers committed to improving organ preservation have been investigating new preservation solutions, machine perfusion, and new preservation techniques.
The further reduction in the temperature means that the metabolic rate is decreased further, which would allow extended preservation time and improved preservation quality. Extreme low-temperature cryopreservation, usually in liquid nitrogen, will cause the cessation of all biochemical activities and permit indefinite storage. However, at present, cryopreservation can only be applied to single cells and some simple tissues, and most complex multicellular tissues and organs do not tolerate cryoinjury.9 Supercooling preservation is another form of lower temperature preservation that diminishes the injuries caused by ice formation at subzero temperatures. Researchers have achieved promising organ preservation results utilizing different supercooling techniques in animal experiments.10-12 Of note, Berendsen et al13 showed that supercooling preservation of the rat liver at –6°C allowed for safe preservation for 72 hours with long-term survival. However, in the field of heart preservation, no encouraging results have been reported, although only short-term supercooling preservation has been evaluated.14-16
We developed a novel supercooling technique using a liquid cooling apparatus and novel preservation and perfusion solutions, which yielded a stable –8°C supercooling state. The aim of our study is to evaluate the preservation effect of our supercooling preservation technique in a mouse heart transplantation model.
MATERIALS AND METHODS
Specific pathogen-free inbred male C3H/HeSlc (C3H) mice (aged 7–10 wk) were purchased from Japan SLC, Inc. (Shizuoka, Japan). All mice received humane care in accordance with the guidelines of the Animal Use and Care Committee of the National Research Institute for Child Health and Development, Tokyo, Japan (Permission number: A2008-004-C10). All mouse experiments conform to the National Institutes of Health guidelines for the care and use of laboratory animals.
A supercooling state was achieved by using a liquid cooling apparatus (Technican Co., Ltd, Kanagawa, Japan), novel preservation, and perfusion solutions (Figure S1, SDC, http://links.lww.com/TP/B855). The liquid cooling apparatus enabled immersed cardiac grafts to be simply, stably, and safely preserved in a nonfreezing supercooling state at –8°C. Novel preservation and perfusion solutions were developed for the supercooling preservation of cardiac grafts. The preservation solution is a hyperosmolar solution modified from Institut Georges Lopez-1 (IGL-1) solution chiefly by employing 35-kDa polyethylene glycol (PEG; Sigma, St. Louis, Missouri), glucose (Sigma), and trehalose (Sigma) for cryoprotection and lidocaine (Sigma) for cardioplegia and myocardial protection (Table 1). The perfusion solution is a moderate osmolality solution that is made by subtracting some components from the preservation solution. After donation, the heart graft was flushed with 10 mL of preservation solution at a rate of 1 mL/min, then placed in a sealable bag filled with 15 mL of preservation solution. The bag was sealed and immersed in the coolant of the liquid cooling apparatus at –8°C for different periods of storage time. At the end of storage, the bag was transferred into an ice–water mixture for rewarming. Before transplantation, the heart graft was perfused with 10 mL of perfusion solution at a rate of 1 mL/min. The intrabag preservation solution was gradually cooled to –8°C at a rate of < 1°C/min, stably maintained, and then gradually rewarmed to 0°C (<1°C/min) (Figure 1). The intrabag temperature was measured by a temperature logger (Thermochron type-SL, DS1922L; KN Laboratories, Inc., Osaka, Japan).
Syngeneic heterotopic heart transplantation was performed as previously described.17-19 After donation, intracardiac blood was flushed out with 10 mL of preservation solution at a rate of 1 mL/min. The cardiac grafts were preserved by supercooling preservation at –8°C or conventional UW preservation at 4°C (Belzer, Bridge to Life Ltd., Columbia, SC) preservation for different periods of time or transplanted immediately as nonpreservation controls. Before transplantation, hyperkalemic UW solution was flushed out with 10 mL of saline, whereas hyperosmolar supercooling preservation solution was flushed out with 10 mL of supercooling perfusion solution, both at a rate of 1 mL/min. For implantation, the warm ischemia time was standardized at 20 minutes. The maximal preservation time was investigated. Twenty-four-hour sample data were collected and analyzed to compare the heart graft protection effect of supercooling preservation at –8°C and conventional UW preservation at 4°C (Figure S2, SDC, http://links.lww.com/TP/B855). The mice were euthanized by CO2 inhalation.
Rebeating Time and Graft Survival
The rebeating time was recorded as the time between aorta declamping and spontaneous recovery of a regular periodic ventricular beating, which was defined as revive. Graft survival was monitored by daily abdominal palpation of the cardiac impulse. Total cessation of heartbeat was confirmed by direct laparotomy.
Measurement of the Tissue Adenosine Triphosphate Level
After 24-hour preservation, the cardiac grafts were immediately immersed in liquid nitrogen (–196°C) and stored frozen at –80°C until used in biochemical analysis. The Adenosine Triphosphate (ATP) levels were measured by an ATP assay kit (ab83355; Abcam, Cambridge, UK), according to the manufacturer’s instructions and expressed as micromoles per gram of tissue.
The Measurement of Myocardial Injury Enzymes
Serum was collected from recipient mice at postoperative hour 24 (POH24) after 24-hour preservation. The serum creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) levels were measured using a chemical analysis system (FUJIFILM DRI-CHEM 3500i; Fujifilm, Tokyo, Japan).
Cardiac grafts were procured at POH24 after 24-hour preservation and, at the end of 24-hour preservation without transplantation, fixed in 10% neutral buffered formalin (WAKO, Osaka, Japan) and then embedded in paraffin. Sections (thickness: 4 μm) were stained with hematoxylin and eosin stain. The perivascular and vascular areas were measured using the Fiji software program.20 The extent of perivascular edema was calculated by subtracting the vascular area from the total area of perivascular edema and then dividing the result by the vascular area.21
Supplementary Materials and Methods
Additional materials and methods including tissue water content, RNA preparation, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry, lipid oxidation assay, tissue infiltrating neutrophil staining, myocardial glycogen staining, and transmission electron microscopy are available in the Supplementary Materials and Methods (SDC, http://links.lww.com/TP/B855). The sequences of the primers and probes used in qRT-PCR are shown in Table S1 (SDC, http://links.lww.com/TP/B855).
The results are expressed as mean ± SD. All data were analyzed using the GraphPad Prism software program (version 7.0; GraphPad Software, San Diego, CA). A 1-way ANOVA and Tukey test were used to compare multiple groups. Heart graft survival was analyzed using Kaplan–Meier curves and log-rank tests. P value of <0.05 was considered to indicate statistical significance.
Preservation Time, Rebeating Time, and Myocardial Injury
Cardiac grafts can be preserved up to 96 hours with long-term survival using our supercooling preservation technique (n = 6 for each group). Even after 144 hours of preservation, cardiac grafts were successfully revived without long-term survival in the supercooling preservation group. However, no heart graft survived after 48-hour conventional 4°C UW preservation. All cardiac grafts achieved long-term survival after 24-hour supercooling preservation and 24-hour conventional UW preservation. The Kaplan–Meier curve analysis showed that the supercooling preservation group had superior survival in comparison to the conventional UW group (Figure 2A) (48 h, P < 0.0001).
The time to spontaneous recovery of heart beating after transplantation in the supercooling preservation group was significantly shorter in comparison to the conventional UW preservation group (Figure 2B) (24-h, P < 0.0001; 48-h, P < 0.0001; n = 9~16 in nonpreservation, UW 24-h, UW 48-h, supercooling 24-h, supercooling 48-h and supercooling 96-h groups; n = 6 in supercooling 120-h and supercooling 144-h groups).
CPK and LDH are important biomarkers of myocardial injury. At POH24 after 24-hour preservation, the serum CPK and LDH levels in the supercooling group (CPK: 172.7 ± 31.35 U/L; LDH: 1079 ± 114.5 U/L) were both significantly lower in comparison to the conventional UW group (CPK: 335.8 ± 39.73 U/L, P < 0.0001, Figure 2C; LDH: 1990 ± 458.3 U/L, P = 0.0001, Figure 2D; n = 6 for each group).
Tissue ATP Levels and Glycogen in Cardiac Grafts
ATP is the primary energy source for all cellular activities. During ex vivo preservation, the pivotal event is rapid ATP depletion following oxygen deprivation. After this, cells shift from aerobic metabolism to anaerobic metabolism, in which only glucose and glycogen can be used as fuel. The poverty of ATP leads to a cascade of cellular edema, intracellular acidosis, and membrane injury, eventually culminating in cell death.22,23 The preserved heart graft tissue ATP concentration (Figure 3A) in the supercooling group (1.781 ± 0.2598 μmol/g) was significantly higher than that in the conventional UW group (0.7604 ± 0.1386 μmol/g, P < 0.0001; n = 6 for each group) after 24-hour preservation. Correspondingly, the periodic acid–Schiff staining also indicated that myocardial glycogen (Figure 3B) was better preserved in the supercooling group than in the conventional UW group after 24-hour preservation (n = 4 for each group).
Myocardial edema is thought to be a generic component of the tissue response to ischemia–reperfusion injury and an important diagnostic marker for assessing the acuity of tissue damage.24 Hematoxylin–eosin staining of heart graft sections showed that the perivascular edema (Figure 4A) in the conventional UW group (1.044 ± 0.2816) was more severe than that in the supercooling group (0.4022 ± 0.08635, P < 0.0001; n = 6 for each group) at POH24 after 24-hour preservation. The myocardial tissue water content (Figure 4B) at postoperative hour (POH) 2 in the supercooling group (76.19 ± 0.9595) was also significantly lower than that in the conventional UW group (78.36 ± 0.8969, P = 0.0017; n = 6 for each group) after 24-hour preservation.
Heart graft mRNA levels of proinflammatory mediators were detected using POH 6 samples after 24-hour preservation (n = 3 in the nonpreservation group, n = 4 in the conventional UW group, n = 5 in the supercooling group) (Figure 5A). The qRT-PCR results showed that the mRNA expression levels of tumor necrosis factor-α, interleukin-1β, and C-C motif chemokine ligand 2 in the supercooling group were significantly decreased in comparison to the conventional UW group. In addition, the mRNA expression levels of interleukin-6, inducible nitric oxide synthase, and interferon-γ in the supercooling group tended to be lower than those in the conventional UW group but did not reach statistical significance. The heme oxygenase 1 and hypoxia-inducible factor 1α mRNA expression levels in the conventional UW group were significantly higher in comparison to the supercooling group, which was presumed to be a result of intrinsic compensatory upregulation of cellular antioxidants. Correlated with these proinflammatory mRNA levels, the numbers of heart graft tissue infiltrating neutrophils in the supercooling group (7.733 ± 2.852), as indicated by naphthol 3-Hydroxy-2-naphthoic-o-toluidide (AS-D) chloroacetate esterase staining (Figure 5B), were significantly lower in comparison to the conventional UW group (26.57 ± 4.847, P < 0.0001; n = 6 for each group).
Oxidative Stress-related Damage
8-Hydroxydeoxyguanosine, an oxidized derivative of deoxyguanosine, is a useful marker for estimating the DNA damage induced by oxidative stress. The level of thiobarbituric acid reactive substances, a byproduct of lipid peroxidation, is considered to be an index of oxidative stress and cellular injury. There were significantly fewer 8-hydroxydeoxyguanosine-positive myocardial cells in the supercooling group (6.540 ± 1.365) in comparison to the conventional UW group (17.70 ± 3.664, P < 0.0001; n = 5 for each group) at POH24 after 24-hour preservation (Figure 6A). The thiobarbituric acid reactive substance level in the supercooling group (3.405 ± 0.4234 μM) was also significantly lower in comparison to the conventional UW group (5.084 ± 0.3873 μM, P < 0.0001; n = 6 for each group) at POH24 after 24-hour preservation (Figure 6B).
Myocardial Cell Apoptosis
Cell apoptosis is an important process leading to cell death caused by myocardial cell ischemia–reperfusion injury. A terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling assay was performed to detect myocardial cell apoptosis at POH24. There were significantly fewer terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling–positive myocardial cells in the supercooling group (3.174% ± 0.6128%) in comparison to the conventional UW group (15.66% ± 2.941%, P < 0.0001; n = 5 for each group) at POH24 after 24-hour preservation (Figure 6C). Caspase-3 has been identified as a key proapoptotic protein involved in the execution phase of multiple apoptotic pathways. There were significantly fewer caspase-3-positive myocardial cells in the supercooling group (5.93 ± 1.72) in comparison to the conventional UW group (14.86 ± 2.411, P < 0.0001; n = 5 for each group) at POH24 after 24-hour preservation (Figure 6D).
The myocardial ultrastructural changes were examined by transmission electron microscopy. After 24-hour preservation in the conventional UW group, mild mitochondrial edema and matrix condensation, contracted sarcomeres, and irregular Z lines could be observed (Figure 7A), while the myocardial ultrastructure was well preserved in the supercooling preservation group (Figure 7B). At POH2, massive swelling mitochondria and sarcomere destruction could be observed in the conventional UW group (Figure 7C). Though a few mitochondria displayed damaged cristae, there was no obvious disruption of the outer mitochondrial membrane in the supercooling preservation group (Figure 7D). The mitochondria score (UW 24-h, 2.670 ± 0.192, supercooling 24-h, 3.788 ± 0.115, P < 0.0001; UW 24-h POH2, 1.288 ± 0.117, supercooling 24-h POH2, 3.503 ± 0.213, P < 0.0001; n = 4 for each group) and sarcomere score (UW 24-h, 1.900 ± 0.161, supercooling 24-h, 2.878 ± 0.077, P < 0.0001; UW 24-h POH2, 1.150 ± 0.048, supercooling 24-h POH2, 2.798 ± 0.103, P < 0.0001; n = 4 for each group) were significantly higher in the supercooling group compared to the conventional UW group (Figure 7E).
To our knowledge, this study showed, for the first time, that our supercooling preservation technique enabled cardiac graft revival after 144-hour preservation, long-term survival after 96-hour preservation, and improved posttransplant outcomes in a mouse heart transplantation model. We lowered the temperature of supercooling heart preservation to –8°C for the first time. Amir et al preserved a heart at –1.3°C using antifreeze protein as a UW additive.16 Seguchi et al realized heart supercooling preservation at –3°C using variable magnetic field technique.25 Our supercooling preservation was achieved using our novel solutions and by simple immersion in the –8°C coolant of the liquid cooling apparatus. This technique may offer an alternative method for clinical organ preservation with a prolonged preservation time and a better preservation effect and is almost as cost-efficient and convenient as conventional UW preservation.
We hold that the –8°C stable supercooling state is the key factor in the improved preservation. Unlike the liver and kidney, the heart is much more vulnerable to ischemic injury. Pumping the circulation system makes the heart consume the most energy per mass unit of any organ in the body.26 It contains large quantities of actin–myosin complexes and mitochondria, which are involved in the contractile function. When ischemic, cardiac ATP is rapidly depleted by catabolic action, leading to lethal contracture injury, oxidative damage, and edema. In comparison to other donor organs, preservation of the heart is highly ATP dependent.27 The fundamental basis for low-temperature preservation is that a low temperature depresses the biochemical activities and energy demand of the donor organ.8 Our supercooling preservation technique preserves heart grafts at –8°C without freezing, which is highly advantageous for preserving the heart in comparison to conventional preservation at 4°C.
Reducing cold-induced membrane injury and avoiding ice formation are the major challenges of supercooling preservation.28,29 To address these challenges, we developed a novel preservation solution modified from IGL-1 solution, which is derived from UW solution.30 The major modifications involved the adaptation of PEG, glucose, trehalose, and lidocaine. Thirty-five-kilo Dalton PEG is an original component of IGL-1 as a nonpermeable colloid, which has been shown to protect the epithelial cell membrane, reduce reactive oxygen species, prevent cell death, maintain mitochondrial integrity, and reduce inflammation and endoplasmic reticulum stress.31-36 We used an increased concentration of PEG as a cryoprotectant.37 We also used high-concentration glucose as a cryoprotectant, as it has been observed to allow freeze-tolerant frogs to resist freezing temperatures as low as –16°C.38,39 We increased the insulin concentration to facilitate the myocardial glucose and potassium uptake and boost glycogen synthesis to prevent cold injury. In addition, the combined use of glucose, insulin, and potassium, namely glucose–insulin–potassium, is generally applied in clinical practices to protect against myocardial ischemia–reperfusion injury and protect the myocardium.40,41 Trehalose is a nontoxic disaccharide of glucose that has been widely used as a natural cryoprotectant for the cryopreservation of cells and human tissues.42-44 Lidocaine, a sodium channel blocker, was introduced for cardioplegia.45 Moreover, the combined use of adenosine, lidocaine, and magnesium has been demonstrated to have cardioprotective and resuscitative properties by leading to a hibernation-like state, reducing inflammation, and lowering the energy demand.46,47 To prepare a hyperosmolar preservation solution, the components of the perfusion solution were reduced to prevent a sudden change of osmotic pressure during the reperfusion phase. We completely avoided ice formation using our novel preservation solution and liquid cooling apparatus.
Under supercooling preservation at –8°C, the myocardial tissue ATP levels were greatly preserved in comparison to the conventional UW preservation group. ATP is the fuel that maintains cell homeostasis and prevents irreversible damage to the organ during hypoxic storage.48 The depletion of ATP stimulated the resynthesis of ATP via aerobic metabolism, which rapidly leads to acidosis and necrosis of the myocardium.49 In the supercooling preservation group, the well-preserved myocardial glycogen suggested that anaerobic metabolism is well suppressed. Moreover, without ATP, the actinomycin cross-bridges are unable to be broken, which leads to irreversible myocardial contracture.50
As expected, the posttransplant outcomes in the supercooling preservation group were significantly better than those in the conventional UW preservation group. Long-term survival was achieved after preservation for up to 96 hours in the supercooling preservation group, which was 4 times as long, as was observed in the conventional UW preservation group. Even after 144-hour preservation, cardiac grafts were successfully revived; however, they failed to survive to the next day. The histological and pathological examination of grafts preserved for 24 hours also showed that the outcomes in the supercooling preservation group were superior to those in the conventional UW preservation group, consistent with previous supercooling studies.14-16 In summary, supercooling preservation protected the myocardial structures and reduced myocardial ischemia–reperfusion injury, oxidative stress-related damage, and myocardial cell apoptosis.
Intolerance of ice-induced injury has hindered the application of cryopreservation in organ preservation. Subzero supercooling preservation with the avoidance of freezing offers a new concept of lower temperature storage as an alternative to conventional preservation at 4°C. Even a minor reduction in temperature may yield markedly improved preservation.51 Our study and recent studies have shown that supercooling preservation appears to be a promising and technically feasible approach for clinical translation in the short term.52 The realization of these efforts may lead to fewer organs being discarded and facilitate global organ sharing and perfect surgical preparation.
Our results clearly showed that supercooling preservation protected cardiac grafts, prolonging the cold storage time and improving posttransplant outcomes in a heterotopic mouse heart transplantation model. However, there are some limitations because of the use of the animal model in our study. Since the mouse heart is a relatively small organ, supercooling preservation should be examined and modified in large animal transplantation before beginning clinical research. The heart graft in a heterotopic transplantation model beats without the workload of supporting a body’s circulation. Myocardial injury is likely to be more severe if evaluated in an orthotopic heart transplantation model, which better mimics clinical heart transplantation. In experimental model, the donor hearts were retrieved from healthy, anesthetized animals, whereas human hearts are obtained from brain dead donors with various conditions. All of the above factors may contribute to differences between experimental and clinical studies. Further studies should be performed before supercooling preservation is applied in clinical practice.
In conclusion, our study indicated that supercooling preservation of mouse cardiac grafts at –8°C greatly prolonged the preservation time and improved posttransplant outcomes in comparison to conventional UW preservation at 4°C. These findings can be explained by the better preservation of ATP levels at lower temperatures. Our study suggests that the development of supercooling preservation techniques will improve the availability of organs and lead to better posttransplant outcomes.
The authors gratefully acknowledge Ms. Sasaki for her technical assistance.
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