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Ann Thorac Surg 2004;77:1648-1655
© 2004 The Society of Thoracic Surgeons
a Heart Transplantation Unit, Tel Hashomer, Israel
b Department of Cardiac Surgery, Sheba Medical Center, Tel Hashomer, Israel
c Department of Biomechanical Engineering, University of California, Berkeley, California, USA
d The Neufeld Cardiac Research Institute, Tel Aviv University, Tel Aviv, Israel
Accepted for publication April 25, 2003.
* Address reprint requests to Dr Amir, Heart Transplantation Unit, Department of Cardiac Surgery, Sheba Medical Center, Tel Hashomer 52621, Israel
e-mail: gabiamir{at}yahoo.com
Presented at the Thirty-ninth Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 31–Feb 2, 2003.
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Abstract |
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METHODS: Forty-two heterotopic transplantations were performed in isoimmune Sprague-Dawley rats. Harvested hearts were retrogradely infused with cold 4°C University of Wisconsin (UW) solution and were preserved in a specialized cooling bath at two target temperatures, 4°C and –1.3°C for 12,18, and 24 hours (6 experiments/group). Preservation solutions were UW alone for the 4°C group, and UW with 15 mg/mL AFP III for the –1.3°C group. After hypothermic storage the hearts were heterotopically transplanted into isoimmune rats. Viability was assessed and graded on a scale of 0 to 6 (0 = no contractions to 6 = excellent contractions). Transplanted hearts were then fixed in vivo and were subject to electron microscopy and histopathologic examination.
RESULTS: None of the hearts preserved at –1.3°C in UW/AFP III solution froze. All control hearts preserved at –1.3°C without AFP protection froze and died at reperfusion. Viability of hearts preserved at –1.3°C in UW/AFP III solution was significantly better after 18 hours of preservation, 30 and 60 minutes after reperfusion (median, 5 versus 3 and 6 versus 3, respectively; p < 0.05) and after 24 hours of preservation 30 and 60 minutes after reperfusion (median, 4.5 versus 1.5 and 5 versus 2, respectively; p < 0.05). Histologic and electron microscopy studies demonstrated better myocyte structure and mitochondrial integrity preservation with UW/AFP III solution.
CONCLUSIONS: Antifreeze proteins prevent freezing in subzero cryopreservation of mammalian hearts for transplantation. Subzero preservation prolongs ischemic times and improves posttransplant viability.
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Introduction |
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Subzero cryopreservation has not been studied extensively because at subzero temperatures the preservation solution freezes and causes irreversible damage to the heart. The freezing point of the preservation solution can be lowered either colligatively, by altering the osmolarity of the preservation solution, or noncolligatively using antifreeze proteins (AFPs).
Colligative high subzero cryopreservation of rat hearts has been tested in isolated rat heart Langendorff experiments using various cryoprotective agents: methanol, ethanol, ethylene glycol, propylene glycol [4], and 2,3-butanediol [5]. In these studies, when preservation time was extended to 9 hours, the decay in function was faster than the decay in adenosine triphosphate levels, suggesting that energy was better preserved than function [4]. The toxicity and osmotic stress of these cryoprotective agents has been implicated with the decay of function in prolonged subzero preservation [4, 6].
Polar fish survive subzero temperatures by producing compounds known as thermal hysteresis proteins or AFPs [6–9]. Arctic fish start to produce AFPs once winter begins and the ambient temperature of 0°C is reached; they survive seawater temperatures as low as –1.8°C with the presence of ice crystals without freezing as a result of the protective effect of the AFPs, which lower the freezing temperature of their body fluids without altering its osmolarity. Antifreeze proteins have several unique properties. The first that was discovered is their ability to depress freezing temperature noncolligatively by several orders of magnitude more than what would be expected from their concentration in the organism's plasma. The mechanism of action of AFPs is not well understood, but it is most probably related to their ability to bind to ice crystals and inhibit recrystalization [9].
Preliminary studies in subzero preservation of rat livers were successful [10, 11], but AFPs failed to enhance storage of the isolated rat heart preparation at hypothermic temperatures and increased damage under freezing conditions [12, 13]. Preliminary studies previously performed by our group demonstrated that AFPs prevent freezing in subzero preservation [14], and that hearts preserved at –1.1°C without AFPs and without the introduction of nucleating agents displayed similar viability scores to hearts preserved at –1.1°C with AFP III (unpublished data), thus demonstrating that AFPs have no detrimental or cardioprotective effect other than cryoprotection.
In vivo experiments designed to examine the cryoprotective effect of AFPs on prolonged subzero heart preservation have not been performed. The present study demonstrates improved functional recovery, survival, and structural integrity of heterotopically transplanted rat hearts after 18 and 24 hours of subzero preservation using AFP III, which is derived from the Ocean Pout (Macrozoarces americanus).
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Material and methods |
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The donor rat underwent a midline abdominal and chest incision separating the anterior chest and the diaphragm; the anterior rib cage was hinged, exposing the heart. University of Wisconsin (UW: Vispan, Dupont) cardioplegic solution, with heparin (500 U/10 mL), was administered through the inferior vena cava to arrest and preserve the heart. After harvesting, hearts were preserved either in UW solution at 4°C or in UW solution with AFP III (15 mg/mL) at –1.3°C for 12, 18, and 24 hours.
Antifreeze protein III was derived directly from the blood of the ocean pout (AFP Inc, Waltham, MA). Temperatures were determined according to preliminary experiments in which thermal hysteresis activity of AFP III was evaluated. Antifreeze protein III at concentrations of 10, 15, and 20 mg/mL depresses the freezing temperature of the solution to –1.4°C, –1.7°C, and –1.8°C, respectively. Preservation temperatures were maintained by using a specialized cooling bath (RTE 140 Neslab, Portsmouth, NH), which maintains a fixed temperature with an accuracy of 0.1°C.
Ice was added to the preservation solution of each experiment as a nucleating agent. Six experiments were performed for each preservation solution and for each preservation time and temperature, overall yielding 36 experiments. Control experiments consisted of six hearts preserved for 12 hours at –1.3°C without AFP III.
In the recipient rats, a long abdominal incision exposed the abdominal aorta and the inferior vena cava. The two vessels were exposed from a short segment just below the renal arteries to the point chosen for the anastomosis. Rewarming of the donor heart is passive and occurs while the heart is transplanted at room temperature, similar to the techniques used in clinical heart transplantation. The ascending aorta was anastomosed to the abdominal aorta, and the pulmonary artery was anastomosed to the inferior vena cava. Viability of the transplanted heart was assessed immediately by direct visualization 30 and 60 minutes after reperfusion and was graded on a scale of 0 to 6; 0 = no contractions, 1 = sporadic contractions, 2 = poor contractions, 3 = moderate contractions, 4 = moderate to good contractions, 5 = good contractions, and 6 = excellent contractions. Visual assessment was performed by the principal investigator (G.A.); nevertheless our preliminary work was graded by three independent observers, and viability scores were similar without significant difference among these blinded independent observers.
After each experiment the hearts were fixed in vivo while beating using a 2% paraformaldehyde and 2.5% glutaraldehyde solution at room temperature for 1 hour, and postfixed for an additional hour with 1% osmium tetroxide at 4°C, dehydrated in alcohol, and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and photographed using a JEOL 1200EX transmission electron microscope.
Values were expressed as medians for descriptive purposes only. Comparisons of viability scores were made between hearts preserved at 4°C and –1.3°C for each preservation time interval separately, 12 hours,18 hours, and 24 hours (comparisons of two groups of nonparametric variables), using the Mann-Whitney test for nonparametric variables. A p value less than 0.05 was considered significant.
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Results |
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12-hour preservation
There was no statistical difference in viability scores among the six hearts preserved using standard storage techniques for 12 hours at 4°C in UW solution and the six hearts preserved at –1.3°C with AFP III (median, 5.5 versus 4.5 and 6 versus 5, respectively; not significant).
18-hour preservation
Hearts preserved for 18 hours at –1.3°C using AFP III displayed superior viability scores compared with hearts preserved using standard storage techniques at 4°C in UW solution 30 and 60 minutes after reperfusion (median, 5 versus 3 and 6 versus 3, respectively; p < 0.05).
24-hour preservation
Hearts preserved for 24 hours at –1.3°C using AFP III displayed superior viability scores compared with hearts preserved using standard storage techniques at 4°C in UW solution 30 and 60 minutes after reperfusion (median, 4.5 versus 1.5 and 5 versus 2, respectively; p < 0.05). Two of the hearts preserved at 4°C for 24 hours died on reperfusion whereas none of the hearts preserved at –1.3°C using AFP III for the same period of time died. Results are summarized in Table 1
and in Figures 1 and 2.
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Electron microscopy studies
Hearts preserved for 12 hours at 4°C showed a mixture of normally appearing cardiomyocyte ultrastructure with other areas of ultrastructural damage evident by clumping of chromatin in the nucleus, dilated mitochondria with excessive swelling of the matrix, and disruption of cristae (Figs 4A–C).
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Control hearts that were preserved for 12 hours at –1.3°C in UW solution without AFP III and froze displayed severe cardiomyocyte ultrastructural damage; mitochondria were completely destroyed with membrane disruption, severe matrix edema, and vacuolization of the cristae. Sarcomeres were not visible (Figs 4F, 4G).
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Comment |
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The northern and Antarctic eel pout and the wolf fish produce a 6.5- to 14-kDa beta sandwich globular protein called AFP III to survive [16]. Antifreeze protein III reduces the freezing temperature of a solution by –1.2°C, bringing it to –1.8°C (plasma osmolarity is 320 mOsm/L, its freezing temperature is –0.6°C) .
The mechanism of action by which AFPs noncolligatively lower the freezing point of the solution is not well understood, but it is most probably related to their ability to bind to ice crystals and inhibit recrystalization [17].
Cryoprotection of solid organs using AFPs has been performed on rat livers and hearts. Successful isolated rat liver subzero preservation has been performed by Lee and colleagues [10]. Rat livers perfused with antifreeze glycoproteins (AFPGs) in Krebs solution before hypothermic storage had a higher rate of bile production and less enzyme leakage on reperfusion compared with livers not perfused with AFPGs. Whole rat livers frozen to –3°C protected by glycerol and AFPG had increased bile production and less hepatocyte structural damage compared with livers preserved with glycerol alone [11].
Isolated perfused rat heart models demonstrated that subzero preservation improves postischemic functional recovery, preserves myocardial adenine nucleotides during ischemia, and prevents myocardial edema at reperfusion [18]. The Langendorff type experiments mentioned above, in which biochemical enzymatic analysis was performed on the heart after 6 hours of hypothermic preservation at –1°C compared with hearts preserved at 4°C, were limited to studying only the effect of temperature and did not explore the effect of preservation time. Hearts preserved at –1°C were not protected from freezing, and nucleating agents were not introduced into the solution.
Cryopreservation of cardiomyocytes or hearts using AFPs has been unsuccessful. Mugnano and colleagues [12] examined the effect of AFPGs on freezing (–4°C) of cardiomyocytes. With the use of cryomicroscopy, they demonstrated that in the solution frozen without AFPG large blunt crystals were formed excluding most cardiomyocytes from the plane of ice formation. After thawing cells appeared similar to unfrozen cells. Spicular ice formed rapidly in the 10-mg/mL AFPG solution. The needlelike crystals appeared to penetrate the cardiomyocytes, resulting in intracellular freezing followed by cell lysis.
Wang and colleagues [13] evaluated subzero cryopreservation of rat hearts using the Langendorff in vitro model of working isolated rat hearts. Cardiac explants were preserved using AFPGs at different concentrations at subzero temperatures of –1.4°C. Hearts that were preserved for 3 hours at concentrations of 10 mg/mL AFPG failed to beat on reperfusion. The authors concluded that AFPGs were deleterious to the isolated rat hearts in a dose-dependent manner, exacerbating the damage caused by freezing.
Our study achieved prolonged 24-hour subzero cryopreservation using AFPs in an in vivo heterotopic heart transplantation model. Our in vivo heart transplantation model is unique and superior to the Langendorff in vitro models because it examines the effect of circulating blood elements such as white blood cells and thrombocytes, which are responsible for much of the reperfusion injury. We believe that the main factor that accounts for the success of our preservation experiments is that subzero preservation using AFPs has to be above the freezing temperature of the solution, because once freezing occurs the damage to the organ is exacerbated and preservation fails [12, 13].
Our results clearly demonstrate that AFP III prevents freezing in the presence of nucleating agents at –1.3°C, and that myocyte destruction caused by freezing is prevented. Furthermore, subzero cryopreservation significantly improved functional recovery of hearts preserved at –1.3°C for 18 and 24 hours compared with hearts preserved at 4°C.
Viability of the transplanted hearts was visually assessed and graded on a scale of 0 to 6. Visual assessment is subjective and was performed by the principal investigator (G.A.); nevertheless our preliminary work was graded by three independent observers, and viability scores were similar without significant difference among these blinded independent observers.
Subzero cryopreservation using AFPs may open a new and exciting frontier in the area of organ preservation.
| Dr Rubinsky discloses that he has a financial relationship with AFP, Inc.
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Discussion |
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DR AMIR: We do not flush them with the solution, In the beginning we flushed them with the antifreeze solution and we had worse results. Now we just preserve them in the solution. We flush them with University of Wisconsin (UW) solution and then preserve them in the preservation solution. The main function of the antifreeze protein is that it inhibits ice formation outside the heart, and once ice does not form outside, it does not form inside.
DR DEL NIDO: So the protein concentration inside the cell is enough to prevent ice formation within the cell, is that what happens?
DR AMIR: The antifreeze proteins do not penetrate the cells.
DR DEL NIDO: No, the normal proteins that are inside the cell, because there is a higher concentration, is that what prevents ice forming inside the cell as well?
DR AMIR: Well, for ice to form, it needs a nidus of ice for nucleation. Once you prevent that nidus from forming outside, it will not form inside the cell. The extracellular volume is much larger than the intracellular volume, so ice formation starts there. If ice forms inside the cell, it stays inside the cell, and one cell dies but not the whole organ.
DR DEL NIDO: The other question I have is where do you see this going, because I think other centers have shown that by minor modifications of UW solution, changing the potassium concentration, and so forth, you can actually get rat hearts preserved for 24 hours. Is this an advantage? Do you foresee this going even longer? And if the concept is simply diffusion from the outside, when you are dealing with a much larger heart, how is it going to work?
DR AMIR: Well, this is the first step. We have done several experiments using other strategies and we want to use all the strategies together. We presented at the European Association a paper of subzero preservation using high concentrations of glucose and insulin, in which using both strategies together can have an additive effect; thus temperatures can be lowered even below –1.3°, –1.5°, even –2°C. So far we were able to prolong ischemic times by 30% to 40%. Several types of antifreeze proteins can lower the temperature to –4° and –5°C, so we can try and use those antifreeze proteins and get about twice as much safe ischemic time.
DR MARSHALL L. JACOBS (Philadelphia, PA): You mentioned several prior experiments by other investigators that were not successful. I think John Baldwin and the heart transplant group at Yale reported in 1994 and 1995 similar prolonged preservation using winter flounder protein as the biologic antifreeze. How did your accomplishments differ from what was reported by that group?
DR AMIR: I am not aware of that paper. I know that previous experiments were not successful. In previous experiment the heart froze and the damage caused by freezing was exacerbated. Our work is unique in that it is performed in vivo with the hearts perfused with all blood elements that are responsible for much of the ischemic and reperfusion injury; that was not done before. All other experiments were Langendorff type in vitro experiments.
DR DAVID G. RABKIN (New York, NY): I thought it was a beautiful study, Doctor Amir, and well presented.
I have two questions. The first is that it was not clear to me what the hemodynamic indices that you used were. Second, I was wondering if you would comment on the clinical relevance of doing hemodynamics on a nonworking heart?
DR AMIR: Excuse me, I did not get the last part.
DR RABKIN: I was wondering if you would comment on the relevance of doing hemodynamics on a nonworking heart. This is a heterotopically transplanted heart and consequently there is no volume in the left ventricle so performing ventricular function studies on a nonworking heart would seem of questionable relevance.
DR AMIR: First, the viability score is part of our study limitations. We have done also Langendorff studies in which we have numbers and figures we can compare, and they are similar to our viability score. Our viability scores were individually checked by three independent observers and were similar. The basic and most important factor is whether the heart survives or not because you have seen that hearts that freeze do not survive and hearts that are protected by antifreeze proteins do survive, that is the main point. It is true that this is a nonworking heart and a nonworking model, but the survival of the myocytes is what is important.
Recently we performed an orthotopic heart transplant in a pig in which the heart was preserved for 19 hours at –1.1°C using antifreeze protein III; the heart sustained the pig for 2 hours until it was harvested.
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