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Cold Storage of the Heart with University of Wisconsin Solution and 2,3-Butanedione Monoxime: Langendorff vs Isolated Working Rabbit Heart Model
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CRYOBIOLOGY 33, ARTICLE NO. 0018
178–185 (1996)
Cold Storage of the Heart with University of Wisconsin Solution and 2,3-Butanedione Monoxime: Langendorff vs Isolated Working Rabbit Heart Model SERGEI Y. LOPUKHIN,1 DIRK F. M. PEEK, JAMES H. SOUTHARD, AND FOLKERT O. BELZER Department of Surgery, University of Wisconsin Hospital, Madison, Wisconsin 53792 Currently, for clinical heart preservation with University of Wisconsin (UW) solution the ischemic time is limited to 8 h. The reliable preservation of the heart for 24 h or more would have a dramatic impact on the existing practice of cardiac transplantation. We showed previously [J. Thorac. Cardiovasc. Surg. 107; 764–775 (1994)] that experimentally preservation could be extended to 24–30 h by preventing ischemic contracture of the heart with 2,3-butanedione monoxime (BDM) in the UW solution (UWBDM). This resulted in nearly 100% return of function as tested in the isolated crystalloid-reperfused rabbit heart in the nonworking Langendorff preparation. We have confirmed these results and now have measured the function of hearts stored in UWBDM for 2, 4, 12, and 24 h using the isolated working rabbit heart model. Preservation in UWBDM solution resulted in a biphasic decrease of cardiac output. In the hearts preserved for 2–12 h the decrease of function averaged 20–35% upon reperfusion, and the differences at 2, 4, or 12 h were not significant (analysis of variance p > 0.05). A more pronounced decrease of 64% was obtained after 24 h of cold storage. Hearts preserved for 24 h without BDM were practically nonfunctional. The release of enzymes (creatine kinase and lactate dehydrogenase) followed biphasic pattern similar to that of cardiac output: a small release between 2 and 12 h and larger, significant losses at 24 h. Although we originally proposed that hearts preserved with UWBDM for 24 h were well preserved (Langendorff model), we now show that poor function was obtained at 24 h. The difference was that in this study we used a more rigorous, isolated working rabbit heart model to test the function of the preserved heart, and this may be a better test of preservation quality. © 1996 Academic Press, Inc.
The University of Wisconsin solution (UW solution) is currently used in some transplant centers for heart preservation (1, 6, 12). Preservation times are limited by these centers to less than 8 h. Because of this short period of acceptable heart preservation there is only a limited area from which a transplant center can depend for a source of cadaveric hearts (about a 1,500km radius from the transplant center). Thus, many cadaveric hearts may be wasted, and some potential recipients who are in need of a heart transplant may die while waiting for an appropriate donor heart. This may be due to our current inability to preserve the heart for 1–2 days, time needed for effective utilization of all cadaveric hearts suitable for transplantation. Preservation of livers, kidneys, or pancreases for 48 h or more has been demonstrated in the
Received April 4, 1995; accepted September 24, 1995. 1 To whom correspondence should be addressed. 178 0011-2240/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
laboratory (4, 9) and in clinics (1). This was accomplished by the development of the UW solution (17). Why this solution is not as effective for heart preservation as for preservation of intraabdominal organs is not known. We proposed earlier that successful heart preservation was limited by ischemic contracture resulting from the loss of ATP from the myocytes. We showed that there was a correlation between the rate of onset of ischemic contracture and the loss of ATP in rabbit hearts (15). Furthermore, we showed that butanedione monoxime (BDM) suppressed the onset of ischemic contracture in rabbit hearts for at least 24 h of cold storage in UW solution, suppressed the rate of loss of ATP and glycogen, and gave nearly 100% return of cardiac function on reperfusion (13, 14). However, in our previous studies, cardiac function was measured in the Langendorff, or nonworking, model, which may not represent accurately the work required by the transplanted heart to support the circulation of
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the recipient (2). In this study we have used a working model to assess how well rabbit hearts were preserved with UW and BDM. We feel that this model is a more rigorous test of preservation quality than the nonworking, Langendorff model. We show that, unlike the results obtained with the Langendorff model, with the working model, rabbit hearts are not well preserved in the UW solution with BDM after 24 h of cold storage. MATERIALS AND METHODS
All animals were treated in compliance with the “Principles of Laboratory Animal Care” formulated by the National Academy of sciences and published by the National Institute of Health (NIH Publication 80-23, revised 1985). New Zealand White rabbits weighing 2 ± 0.2 kg were anesthetized with intravenous sodium thiopental, heparinized (1,000 U intravenously), paralyzed with succinylcholine (10 mg intravenously), and mechanically ventilated via tracheostomy (Harvard rodent ventilator). A median sternotomy was performed, and the heart was arrested by a bolus injection of KCl (4 mEq intravenously), excised, and immersed in icecold saline. The aorta was cannulated and the heart either used immediately (control heart) or flushed with 60 ml of preservation solution at 4°C from a height of 80 cm and stored in a container filled with preservative surrounded with slushed ice in a refrigerated room (4°C). The function of the heart was tested by using either a working model or Langendorff reperfusion method as described elsewhere (2, 14). Working Model In the working model, a cannula was placed in the left atrium and secured with a pursestring suture. The hearts were initially perfused for 15 min on a recirculating Langendorff circuit at a perfusion pressure of 80 cmH2O (the first 100 ml of perfusate passing through the heart was discarded to remove blood elements or preservative) and then switched to a working circuit for the 105-min test period. Each recirculating system was primed with 300 ml of filtered (0.45
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m, Millipore) Krebs–Henseleit solution at 37°C which was equilibrated with 95% oxygen and 5% carbon dioxide (PO2 4 600 torr). The left atrial filling pressure (preload) was set at 20 cmH2O, and the heart was pumping against the column of fluid with overflow set at 80 cmH2O (afterload). A 20-ml compliance chamber was included in the circuit. Aortic pressure was measured continuously at the level of the aortic cannula with a Statham pressure transducer connected to a Gilson ICT-1H chart-recorder. Coronary and aortic flows (CF and AF) were measured every 15 min by timed collections of effluent. Cardiac output (CO), AF, and CF were calculated in ml/g of initial heart weight/min. The hearts were weighed before and after reperfusion, and the weight gain (WG) was calculated as the percent of initial weight. The hearts did not gain weight during cold storage. Nonworking Model The method is identical to the one described by Stringham et al. (14). The hearts were reperfused at 80 cmH2O for 90 min. Systolic pressure was isovolumetrically measured every 15 min with a fluid-filled latex balloon placed in the left ventricle. The balloon was connected to a Statham pressure transducer with Gilson ICT1H chart recorder and was inflated to a diastolic pressure of 20 mmHg. The balloon was deflated between measurements. The system contained 300 ml of oxygenated recirculating Krebs– Henseleit perfusate (PO2 > 600 mmHg) at 37°C (first 100 ml of perfusate was discarded). The CF was measured by timed collections of the effluent. Left ventricular developed pressure (LVDP) was calculated by subtracting the enddiastolic pressure from systolic pressure. Measurements of Enzymes and High Energy Phosphate Compounds Lactate dehydrogenase (LDH) and creatine kinase (CK) concentrations in the perfusate were measured immediately at the end of reperfusion using a Kodak Ektachem DT-60 analyzer and expressed in units/liter. These values were then divided by the initial weight of the heart. In
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separate experiments at the end of the storage period tissue samples were freeze-clamped and processed for the high performance liquid chromatography (HPLC) separation and quantitation of high energy phosphates as described previously (16). The samples were homogenized in 7% perchloric acid and centrifuged at 4,470 g for 10 min. The supernatant was neutralized with 2.4 M Trizma base and centrifuge-filtered through 0.45-m Millipore filters. Analysis for CP, AMP, ADP, and ATP was performed with isocratic HPLC using Shimadzu SPD-6-AV spectrophotometer at wavelength 206 nm, Spectra-Physics Iso-Chrom LC pump set at 1 ml/min flow rate and reverse-phase Supelcosil 15-cm column as described by Sellevold et al. (11). The pellet was resuspended in 10% deoxycholic acid and analyzed for protein content by the Biuret method on a Shimadzu UV-160 spectrophotometer. Results were expressed as nmol of nucleotide/mg of protein. Each sample was processed in duplicate and the mean calculated. Solutions Solutions were prepared and filtered (0.2-m
Millipore) ex tempore in the laboratory. The composition of solutions is shown in Table 1. Experimental Groups Hearts immediately reperfused and functionally evaluated (without preservative flushout) were used as a control group. The experimental groups were hearts exposed to 2, 4, 12, and 24 h of cold storage with UW solution containing 30 mM BDM (UWBDM). Some hearts were evaluated after 24-h cold storage in UW solution without BDM. In the nonworking model there were two groups: control hearts, evaluated immediately after the harvest without flushout with preservative; and hearts cold stored for 24 h in UWBDM solution. The number of hearts in each group is shown in the tables. Statistical Analysis Statistical analysis of data was performed using analysis of variance (ANOVA) with Tukey– Kremer test. Results are expressed as means ± SEM. Values obtained during reperfusion
TABLE 1 Solutions Used in the Study Krebs–Henseleit perfusate NaHCO3 KH2PO4 NaCl KCl MgSO4 CaCl2 Glucose Insulin
24.9 mM 1.2 mM 118.0 mM 4.7 mM 1.1 mM 2.0 mM 10.0 mM 10 U/l
University of Wisconsin solution Lactobionic acid KOH NaOH Adenosine Allopurinol KH2PO4 MgSO4 Raffinose Glutathione Hydroxyethyl starch Dexamethasone Insulin Heparin Sulfamethoxazole Trimethoprime BDMa CaCl2a
Note. Solutions were prepared and filtered (0.2 m Millipore) ex tempore in the laboratory. This chemical was added to prepare UWBDM solution.
a
100.0 mM 100.0 mM 40.0 mM 5.0 mM 1.0 mM 25.0 mM 5.0 mM 30.3 mM 3.0 mM 50 g/l 8.0 mg/l 40.0 U/l 5000.0 U/l 160.0 mg/l 32.0 mg/l 30.0 mM 1.0 mM
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within each group were compared by repeated measures ANOVA with significance taken as p < 0.05. RESULTS
In a previous study Stringham et al. (14) showed that rabbit hearts preserved for 24 h in UWBDM functioned nearly as well as control hearts when evaluated by isolated perfusion in the Langendorff model with LVDP used as the criterion for function. We have repeated this study using six rabbit hearts in the control group and six in the 24-h UWBDM group and obtained similar results. LVDP was 108 ± 6 mmHg in the control group and 111 ± 7 mmHg in the preserved group, after 90 min of reperfusion in the Langendorff model (Table 2). There were no differences in other parameters measured: CF, CK, and LDH release. In the working model we used CO as a measure of cardiac function. CO remained relatively stable for 120 min of reperfusion of fresh and preserved hearts (no significant change). In control hearts CO was 42.3 ± 2.2 ml/min/g after 30 min of reperfusion and 37.6 ± 3.0 ml/min/g after 120 min of reperfusion. The results in Table 3 compare CO in hearts preserved for up to 24 h with control hearts using the results obtained after 120-min reperfusion. Similar results were obtained if CO after 30 or 60 min of reperfusion was compared between groups. Preservation in UWBDM resulted in a biphasic decrease in CO. The first phase occurred in hearts preserved for 2–12 h and was characterized by an approximately 20–35% decrease in CO. The CO values at 2, 4, and 12 hr were 78,
TABLE 3 Cardiac Output after 120 min of Reperfusion ml/min/g Control 2h 4h 12 h 24 h 24 h no BDM
a
37.6 ± 3.0 29.3 ± 2.7a 29.6 ± 1.8a 24.9 ± 4.3a,b 13.7 ± 1.0 52 ± 0.2c
% of control
n
100 78 ± 7 79 ± 5 66 ± 11 36 ± 3 14 ± 1
7 6 6 6 11 4
Note. Values are shown represent the mean ± SE for each group. P values were obtained by ANOVA + Tukey– Kramer post test. n, number of hearts in each group; BDM, 2,3-butanedione monoxime (30mM). a P < 0.001 vs 24 hr. b P < 0.001 vs control. c P < 0.001 vs all other groups.
79, and 66% of control hearts and were not significantly different from each other, although the CO value at 12 h was significantly different (p < 0.01) from the control group. The second phase occurred in hearts preserved for 24 h and was characterized by a large decrease in CO to 36% of fresh hearts. The CO at 24 h was significantly less than the CO at 2, 4, or 12 h (p < 0.001) and at 0 h (p < 0.001). Hearts preserved for 24 h in UW solution without BDM showed greater injury than those preserved for the same time with BDM (Table 3). Without BDM CO decreased to 14% of controls, and the hearts showed practically no AF after 120 min of reperfusion. In this study there was no significant effect of preservation on heart rate (expressed as beats/ min), which in controls was 192 ± 5 and 206 ± 4, 195 ± 9, 218 ± 10, 227 ± 5, and 202 ± 19 for
TABLE 2 Langendorff Model Data
Control 24-h UWBDM
DP (mmHg)
CF (ml/min/g)
CK (U/l/g)
LDH (U/l/g)
n
108 ± 6 111 ± 7
10.0 ± 0.5 7.4 ± 0.9
142 ± 17 150 ± 24
108 ± 5 137 ± 33a
6 6
Note. Data were obtained after 90 min of reperfusion. Values shown represent the mean ± SE for each group. p values were obtained by t test. a p < 0.001 vs control. DP, developed pressure; CF, coronary flow; CK, creatine kinase concentration; LDH, lactate dehydrogenase concentration in the perfusate; UWBDM, University of Wisconsin solution modified with 2,3-butanedione monoxime and 1 mM Ca2+; n 4 number of hearts in each group.
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hearts preserved for 2, 4, 12, 24, and 24 h (no BDM), respectively. All hearts gained from 47 to 64% weight after 120 min reperfusion, and there were no significant differences between groups. Furthermore, the WG was not related directly to preservation time. Control hearts gained 57 ± 7%, while those preserved for 12 h gained 46 ± 3%, and those for 24 h gained 49 ± 6%. The effects of preservation time on enzyme release (CK and LDH) are shown in Fig. 1. The release of enzymes appeared to show a biphasic effect dependent upon preservation time, which was similar to the effect of preservation time on CO. In hearts preserved for 2, 4, or 12 h the amount of CK or LDH released from the heart was not significantly different from the amount released from fresh hearts during 120 min of reperfusion. However, after 24-h preservation there was a sharp increase in the amount of CK released and a smaller increase in LDH release compared with hearts preserved for 12 h or less. The amounts of CK and LDH in the perfusate from 24-h preserved hearts were significantly greater than those released by fresh hearts.
However, the amounts of enzymes (CK and LDH) in the perfusate from hearts preserved for 2, 4, or 12 h were not significantly greater than the amounts in the perfusate from a freshly perfused heart. Hearts preserved for 24 h without BDM showed LDH and CK release similar to that of hearts preserved for 24 h with BDM, and the differences (±BDM) were not significant at p > 0.05. The results in Table 4 show how preservation with or without BDM for 24 h affected high energy phosphate metabolites in the heart after cold storage. In hearts preserved with BDM, creatine phosphate content was unmeasurable. However, the ATP content was similar to fresh hearts (not significantly different) as was the total adenine nucleotide content. Because the ATP content after 24-h storage with BDM was similar to control hearts we did not measure the concentrations of high energy phosphate after shorter periods of preservation. We presumed that, after shorter periods of preservation the high energy phosphate content would not be significantly less than that observed after 24-h preservation.
FIG. 1. Creatine kinase (CK) and lactate dehydrogenase (LDH) concentration in perfusate after 120 min or reperfusion. Values reflect the mean ± standard error of the mean for each group. *p < 0.05 vs control; # P < 0.001 vs control.
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COLD STORAGE OF THE HEART TABLE 4 Myocardial High Energy Phosphate Content
Control 24 UWBDM 24 UW
Cr. Ph
AMP
ADP
ATP
TAN
n
6 ± 5.1 0 0
3.8 ± 0.44 4.6 ± 0.23 11.9 ± 3.8a,b
9.8 ± 0.49 11.2 ± 1.1 10.4 ± 2.6
37.0 ± 2.0 33.5 ± 1.3 12.4 ± 3.6a,b
50.0 ± 2.4 49.3 ± 1.8 34.6 ± 7.4a,b
10 12 8
Note. High energy phosphates were assayed by high performance liquid chromatography after cold storage. Values are nm/mg of protein and represent the mean ± standard error for each group. P values were obtained by ANOVA + Tukey–Kramer post test. Cr. Ph., creatine phosphate; AMP, adenosine monophosphate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; TAN, total adenine nucleotides; n, number of hearts in a group. a p < 0.05 vs control. b p < 0.05 vs 24 UWBDM.
Hearts preserved without BDM for 24 h showed a loss of high energy phosphates compared with fresh hearts or those preserved for 24 h with BDM. ATP decreased by 68% compared with control hearts and was significantly less than in hearts preserved for 24 h with BDM. DISCUSSION
The working heart is considered to be a more rigorous test of cardiac function than the Langendorff nonworking heart because the heart pumps perfusate against a hydrostatic pressure rather than against a fluid-filled latex balloon placed in the left ventricle. This may resemble more closely the work that the heart would be required to do after transplantation and represent more closely the work required to maintain the circulation of the recipient. Galinanes and Hearse (2) compared the effects of global ischemia on the recovery of function of rat hearts reperfused by either the Langendorff or working model. They showed that the working model was more rigorous. Although the recovery of function was similar after 30 min of warm ischemia, after 45 min of global ischemia the recovery of function was considerably less in the working model (4%) than in the Langendorff model (34%). Thus, for short periods of injury (such as short periods of preservation or global warm ischemia) either model may be appropriate to measure recovery of cardiac function. For longer periods of injury the working model may be a better test of how well the heart recovers function and may be more relevant to long term
heart preservation studies to assess the functional competence of the heart. This conclusion appears to be borne out by the results presented here. We show that the recovery of cardiac function (LVDP) as measured in the Langendorff model after 24-h preservation with UWBDM was nearly 100% (similar to a previous study) but not nearly so good when measured in the working model. After 24-h preservation with UWBDM, recovery of cardiac function (CO) was poor when tested in the working model and equal to only 36% of fresh hearts. From our results we postulate that there are two phases of injury to the heart due to preservation. The first phase is reversible injury, and the second phase is irreversible injury. The initial injury occurs rapidly after flushout with a hyperkalemic, hypothermic preservative such as the UW solution. After only 2 h of cold storage CO is reduced by about 22% of controls. After 4 or 12 h of storage there is no significantly greater decrease in CO compared with the decrease at 2 h. During this initial phase of injury significant increases in cellular damage and increased leakage of intracellular enzymes did not occur. The concentrations of both CK and LDH released into the reperfusion fluid were similar at 2, 4, and 12 h and also similar to fresh hearts. At 24-h preservation, however, there was a significant decrease in CO to 36% of fresh hearts. The CO at 24 h was significantly different than at 2, 4, or 12 h. Also, after 24-h preservation there was a large increase in the amount of CK and LDH released into the reper-
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fusion medium, indicative of cell membrane damage. The increased degree of injury at 24 h does not appear due to a loss of ATP, which is near normal after 24-h storage. There is evidence in the literature which is somewhat supportive of the biphasic nature of injury to the preserved heart. For instance, it is well known that hearts tolerate about 4 h of cold storage, and this is currently the accepted limits for human heart preservation. Recently, clinical heart preservation has been extended in a few cases to 8 h (1). Also, Jeevanandam et al. (5) have shown in the orthotopic heart transplant model using the baboon that hearts preserved for about 12 h in UW solution are fully viable. However, hearts preserved for longer periods were irreversibly injured, and no animal survived after transplantation. The initial injury that occurs with cold flushout of the heart does not appear, therefore, to lead to failure of the transplant. However, it is well known that up to 25% of the preserved hearts transplanted within about 4 h develop serious complications related to preservation/ reperfusion injury (3). Thus, even though the injury may be reversible, it is serious, and methods to suppress this initial injury may be necessary to improve cardiac preservation. Currently, methods to suppress the injury caused by short term preservation involve some form of suppression of reperfusion injury. Drugs such as inotropes, vasodilators, and calcium-regulating agents may be important in hearts preserved for short periods of time. The greater degree of injury occurs in the second phase, after 12-h storage, and what causes this injury and how to suppress it are not known. Hearts preserved for long periods of time may not be amenable to improvement by treating the reperfusion injury with various drugs because the hearts are irreversibly damaged by the long period of cold ischemia. Why hearts do not tolerate preservation greater than about 12 h is not known. Other transplantable organs (liver, kidney, pancreas) are well known to tolerate up to 3 days of preservation in the UW solution. We proposed earlier that one of the limitations to successful long
term heart preservation was ischemic contraction induced by the loss of ATP (14). The loss of ATP in the other transplantable organs is well tolerated, and because of the low concentration of contractile proteins in these organs they do not undergo the type of contracture seen in cardiac muscle. We showed that BDM suppressed ischemic contracture, and we proposed that this agent, or a similar method to suppress ischemic contracture, would be a necessary component of effective long term heart preservation. In this study we show that although CO is depressed after 24-h preservation in UWBDM, without BDM the loss of function after 24-h storage is even greater than with BDM in the UW solution. Thus, BDM may be an important agent in preservatives used for long term heart preservation. However, BDM is not sufficient by itself to extend safe preservation times beyond about 12 h, and other modifications of the preservative or method of preservation will probably be needed to accomplish the goal of truly long term (30–48 h) heart preservation. In summary, this study shows that simple cold storage of the heart with the UWBDM does not appear effective for 24-h preservation when tested in the isolated perfused, working, rabbit heart model. The goal of cardiac preservation is to obtain a time of preservation that will allow effective sharing of hearts on a national level so that no heart is wasted because of lack of sufficient preservation capabilities. For other transplantable organs this period of time is about 24 h. Most livers, kidneys, and pancreases cold stored in UW solution are transplanted within 24 h of harvest, which is sufficient for national sharing. Very few organs are lost because they have been preserved for a period of time that is considered unsafe. Thus, it appears that clinical heart preservation, for optimal utilization of hearts, should be for at least 24 h. To have assurance that the hearts preserved for 24 h would be life sustaining, a demonstration of 30 or more h of successful preservation in the orthotopic transplant model in the laboratory is probably necessary. Current studies indicate that this may be difficult to obtain by simple cold storage. Instead, for successful heart pres-
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ervation the method of choice may be continuous perfusion, which has been shown to give the longest and best quality preservation of the kidney (7), liver (8), and heart (10).
9.
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1. D’Alessandro, A. M., Southard, J. H., Love, R. B., and Belzer, F. O. Organ preservation. Surg. Clin. North Am. 74, 1083–1095, (1994). 2. Galinanes, M., and Hearse, D. J. Assessment of ischemic injury and protective interventions: The Langendorff versus the working rat heart preparation. Can. J. Cardiol. 6, 83–91, (1990). 3. Hauptman, P. J., Aranki, S., Mudge, G. H., Jr., Couper, G. S., and Loh, E. Early cardiac allograft failure after orthotopic heart transplantation. Am. Heart J. 127, 179–186, (1994). 4. Jamieson, N. V., Sundberg, R., Lindell, S., Claesson, K., Moen, J., Vreugdenhil, P. K., Wight, D. G., Southard, J. H., and Belzer, F. O. Preservation of the canine liver for 24–48 hours using simple cold storage with UW solution. Transplantation 46, 517– 522, (1988). 5. Jeevanandam, V., Auteri, J. S., Marboe, C. C., Hsu, D., Sanchez, J. A., and Smith, C. R. Extending the limits of donor heart preservation: A trial with University of Wisconsin solution. Transplant. Proc. 23, 697–698, (1991). 6. Jeevanandam, V., Barr, M. L., Auteri, J. S., Sanchez, J. A., Fong, J., Schenkel, F. A., Michler, R. E., Smith, C. R., and Rose, E. A. University of Wisconsin solution versus crystalloid cardioplegia for human donor heart preservation: A randomized blinded prospective clinical trial. J. Thorac. Cardiovasc. Surg. 103, 194–198, (1992). 7. McAnulty, J. F., Vreugdenhil, P. K., Lindell, S., Southard, J. H., and Belzer, F. O. Successful 7-day perfusion preservation of the canine kidney. Transplant. Proc. 25, 1642–1644, (1993). 8. Pienaar, B. H., Lindell, S. L., Van Gulik, T., Southard, J. H., and Belzer, F. O. Seventy-two-hour preserva-
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CRYOBIOLOGY 33, ARTICLE NO. 0018
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Cold Storage of the Heart with University of Wisconsin Solution and 2,3-Butanedione Monoxime: Langendorff vs Isolated Working Rabbit Heart Model SERGEI Y. LOPUKHIN,1 DIRK F. M. PEEK, JAMES H. SOUTHARD, AND FOLKERT O. BELZER Department of Surgery, University of Wisconsin Hospital, Madison, Wisconsin 53792 Currently, for clinical heart preservation with University of Wisconsin (UW) solution the ischemic time is limited to 8 h. The reliable preservation of the heart for 24 h or more would have a dramatic impact on the existing practice of cardiac transplantation. We showed previously [J. Thorac. Cardiovasc. Surg. 107; 764–775 (1994)] that experimentally preservation could be extended to 24–30 h by preventing ischemic contracture of the heart with 2,3-butanedione monoxime (BDM) in the UW solution (UWBDM). This resulted in nearly 100% return of function as tested in the isolated crystalloid-reperfused rabbit heart in the nonworking Langendorff preparation. We have confirmed these results and now have measured the function of hearts stored in UWBDM for 2, 4, 12, and 24 h using the isolated working rabbit heart model. Preservation in UWBDM solution resulted in a biphasic decrease of cardiac output. In the hearts preserved for 2–12 h the decrease of function averaged 20–35% upon reperfusion, and the differences at 2, 4, or 12 h were not significant (analysis of variance p > 0.05). A more pronounced decrease of 64% was obtained after 24 h of cold storage. Hearts preserved for 24 h without BDM were practically nonfunctional. The release of enzymes (creatine kinase and lactate dehydrogenase) followed biphasic pattern similar to that of cardiac output: a small release between 2 and 12 h and larger, significant losses at 24 h. Although we originally proposed that hearts preserved with UWBDM for 24 h were well preserved (Langendorff model), we now show that poor function was obtained at 24 h. The difference was that in this study we used a more rigorous, isolated working rabbit heart model to test the function of the preserved heart, and this may be a better test of preservation quality. © 1996 Academic Press, Inc.
The University of Wisconsin solution (UW solution) is currently used in some transplant centers for heart preservation (1, 6, 12). Preservation times are limited by these centers to less than 8 h. Because of this short period of acceptable heart preservation there is only a limited area from which a transplant center can depend for a source of cadaveric hearts (about a 1,500km radius from the transplant center). Thus, many cadaveric hearts may be wasted, and some potential recipients who are in need of a heart transplant may die while waiting for an appropriate donor heart. This may be due to our current inability to preserve the heart for 1–2 days, time needed for effective utilization of all cadaveric hearts suitable for transplantation. Preservation of livers, kidneys, or pancreases for 48 h or more has been demonstrated in the
Received April 4, 1995; accepted September 24, 1995. 1 To whom correspondence should be addressed. 178 0011-2240/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
laboratory (4, 9) and in clinics (1). This was accomplished by the development of the UW solution (17). Why this solution is not as effective for heart preservation as for preservation of intraabdominal organs is not known. We proposed earlier that successful heart preservation was limited by ischemic contracture resulting from the loss of ATP from the myocytes. We showed that there was a correlation between the rate of onset of ischemic contracture and the loss of ATP in rabbit hearts (15). Furthermore, we showed that butanedione monoxime (BDM) suppressed the onset of ischemic contracture in rabbit hearts for at least 24 h of cold storage in UW solution, suppressed the rate of loss of ATP and glycogen, and gave nearly 100% return of cardiac function on reperfusion (13, 14). However, in our previous studies, cardiac function was measured in the Langendorff, or nonworking, model, which may not represent accurately the work required by the transplanted heart to support the circulation of
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the recipient (2). In this study we have used a working model to assess how well rabbit hearts were preserved with UW and BDM. We feel that this model is a more rigorous test of preservation quality than the nonworking, Langendorff model. We show that, unlike the results obtained with the Langendorff model, with the working model, rabbit hearts are not well preserved in the UW solution with BDM after 24 h of cold storage. MATERIALS AND METHODS
All animals were treated in compliance with the “Principles of Laboratory Animal Care” formulated by the National Academy of sciences and published by the National Institute of Health (NIH Publication 80-23, revised 1985). New Zealand White rabbits weighing 2 ± 0.2 kg were anesthetized with intravenous sodium thiopental, heparinized (1,000 U intravenously), paralyzed with succinylcholine (10 mg intravenously), and mechanically ventilated via tracheostomy (Harvard rodent ventilator). A median sternotomy was performed, and the heart was arrested by a bolus injection of KCl (4 mEq intravenously), excised, and immersed in icecold saline. The aorta was cannulated and the heart either used immediately (control heart) or flushed with 60 ml of preservation solution at 4°C from a height of 80 cm and stored in a container filled with preservative surrounded with slushed ice in a refrigerated room (4°C). The function of the heart was tested by using either a working model or Langendorff reperfusion method as described elsewhere (2, 14). Working Model In the working model, a cannula was placed in the left atrium and secured with a pursestring suture. The hearts were initially perfused for 15 min on a recirculating Langendorff circuit at a perfusion pressure of 80 cmH2O (the first 100 ml of perfusate passing through the heart was discarded to remove blood elements or preservative) and then switched to a working circuit for the 105-min test period. Each recirculating system was primed with 300 ml of filtered (0.45
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m, Millipore) Krebs–Henseleit solution at 37°C which was equilibrated with 95% oxygen and 5% carbon dioxide (PO2 4 600 torr). The left atrial filling pressure (preload) was set at 20 cmH2O, and the heart was pumping against the column of fluid with overflow set at 80 cmH2O (afterload). A 20-ml compliance chamber was included in the circuit. Aortic pressure was measured continuously at the level of the aortic cannula with a Statham pressure transducer connected to a Gilson ICT-1H chart-recorder. Coronary and aortic flows (CF and AF) were measured every 15 min by timed collections of effluent. Cardiac output (CO), AF, and CF were calculated in ml/g of initial heart weight/min. The hearts were weighed before and after reperfusion, and the weight gain (WG) was calculated as the percent of initial weight. The hearts did not gain weight during cold storage. Nonworking Model The method is identical to the one described by Stringham et al. (14). The hearts were reperfused at 80 cmH2O for 90 min. Systolic pressure was isovolumetrically measured every 15 min with a fluid-filled latex balloon placed in the left ventricle. The balloon was connected to a Statham pressure transducer with Gilson ICT1H chart recorder and was inflated to a diastolic pressure of 20 mmHg. The balloon was deflated between measurements. The system contained 300 ml of oxygenated recirculating Krebs– Henseleit perfusate (PO2 > 600 mmHg) at 37°C (first 100 ml of perfusate was discarded). The CF was measured by timed collections of the effluent. Left ventricular developed pressure (LVDP) was calculated by subtracting the enddiastolic pressure from systolic pressure. Measurements of Enzymes and High Energy Phosphate Compounds Lactate dehydrogenase (LDH) and creatine kinase (CK) concentrations in the perfusate were measured immediately at the end of reperfusion using a Kodak Ektachem DT-60 analyzer and expressed in units/liter. These values were then divided by the initial weight of the heart. In
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separate experiments at the end of the storage period tissue samples were freeze-clamped and processed for the high performance liquid chromatography (HPLC) separation and quantitation of high energy phosphates as described previously (16). The samples were homogenized in 7% perchloric acid and centrifuged at 4,470 g for 10 min. The supernatant was neutralized with 2.4 M Trizma base and centrifuge-filtered through 0.45-m Millipore filters. Analysis for CP, AMP, ADP, and ATP was performed with isocratic HPLC using Shimadzu SPD-6-AV spectrophotometer at wavelength 206 nm, Spectra-Physics Iso-Chrom LC pump set at 1 ml/min flow rate and reverse-phase Supelcosil 15-cm column as described by Sellevold et al. (11). The pellet was resuspended in 10% deoxycholic acid and analyzed for protein content by the Biuret method on a Shimadzu UV-160 spectrophotometer. Results were expressed as nmol of nucleotide/mg of protein. Each sample was processed in duplicate and the mean calculated. Solutions Solutions were prepared and filtered (0.2-m
Millipore) ex tempore in the laboratory. The composition of solutions is shown in Table 1. Experimental Groups Hearts immediately reperfused and functionally evaluated (without preservative flushout) were used as a control group. The experimental groups were hearts exposed to 2, 4, 12, and 24 h of cold storage with UW solution containing 30 mM BDM (UWBDM). Some hearts were evaluated after 24-h cold storage in UW solution without BDM. In the nonworking model there were two groups: control hearts, evaluated immediately after the harvest without flushout with preservative; and hearts cold stored for 24 h in UWBDM solution. The number of hearts in each group is shown in the tables. Statistical Analysis Statistical analysis of data was performed using analysis of variance (ANOVA) with Tukey– Kremer test. Results are expressed as means ± SEM. Values obtained during reperfusion
TABLE 1 Solutions Used in the Study Krebs–Henseleit perfusate NaHCO3 KH2PO4 NaCl KCl MgSO4 CaCl2 Glucose Insulin
24.9 mM 1.2 mM 118.0 mM 4.7 mM 1.1 mM 2.0 mM 10.0 mM 10 U/l
University of Wisconsin solution Lactobionic acid KOH NaOH Adenosine Allopurinol KH2PO4 MgSO4 Raffinose Glutathione Hydroxyethyl starch Dexamethasone Insulin Heparin Sulfamethoxazole Trimethoprime BDMa CaCl2a
Note. Solutions were prepared and filtered (0.2 m Millipore) ex tempore in the laboratory. This chemical was added to prepare UWBDM solution.
a
100.0 mM 100.0 mM 40.0 mM 5.0 mM 1.0 mM 25.0 mM 5.0 mM 30.3 mM 3.0 mM 50 g/l 8.0 mg/l 40.0 U/l 5000.0 U/l 160.0 mg/l 32.0 mg/l 30.0 mM 1.0 mM
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within each group were compared by repeated measures ANOVA with significance taken as p < 0.05. RESULTS
In a previous study Stringham et al. (14) showed that rabbit hearts preserved for 24 h in UWBDM functioned nearly as well as control hearts when evaluated by isolated perfusion in the Langendorff model with LVDP used as the criterion for function. We have repeated this study using six rabbit hearts in the control group and six in the 24-h UWBDM group and obtained similar results. LVDP was 108 ± 6 mmHg in the control group and 111 ± 7 mmHg in the preserved group, after 90 min of reperfusion in the Langendorff model (Table 2). There were no differences in other parameters measured: CF, CK, and LDH release. In the working model we used CO as a measure of cardiac function. CO remained relatively stable for 120 min of reperfusion of fresh and preserved hearts (no significant change). In control hearts CO was 42.3 ± 2.2 ml/min/g after 30 min of reperfusion and 37.6 ± 3.0 ml/min/g after 120 min of reperfusion. The results in Table 3 compare CO in hearts preserved for up to 24 h with control hearts using the results obtained after 120-min reperfusion. Similar results were obtained if CO after 30 or 60 min of reperfusion was compared between groups. Preservation in UWBDM resulted in a biphasic decrease in CO. The first phase occurred in hearts preserved for 2–12 h and was characterized by an approximately 20–35% decrease in CO. The CO values at 2, 4, and 12 hr were 78,
TABLE 3 Cardiac Output after 120 min of Reperfusion ml/min/g Control 2h 4h 12 h 24 h 24 h no BDM
a
37.6 ± 3.0 29.3 ± 2.7a 29.6 ± 1.8a 24.9 ± 4.3a,b 13.7 ± 1.0 52 ± 0.2c
% of control
n
100 78 ± 7 79 ± 5 66 ± 11 36 ± 3 14 ± 1
7 6 6 6 11 4
Note. Values are shown represent the mean ± SE for each group. P values were obtained by ANOVA + Tukey– Kramer post test. n, number of hearts in each group; BDM, 2,3-butanedione monoxime (30mM). a P < 0.001 vs 24 hr. b P < 0.001 vs control. c P < 0.001 vs all other groups.
79, and 66% of control hearts and were not significantly different from each other, although the CO value at 12 h was significantly different (p < 0.01) from the control group. The second phase occurred in hearts preserved for 24 h and was characterized by a large decrease in CO to 36% of fresh hearts. The CO at 24 h was significantly less than the CO at 2, 4, or 12 h (p < 0.001) and at 0 h (p < 0.001). Hearts preserved for 24 h in UW solution without BDM showed greater injury than those preserved for the same time with BDM (Table 3). Without BDM CO decreased to 14% of controls, and the hearts showed practically no AF after 120 min of reperfusion. In this study there was no significant effect of preservation on heart rate (expressed as beats/ min), which in controls was 192 ± 5 and 206 ± 4, 195 ± 9, 218 ± 10, 227 ± 5, and 202 ± 19 for
TABLE 2 Langendorff Model Data
Control 24-h UWBDM
DP (mmHg)
CF (ml/min/g)
CK (U/l/g)
LDH (U/l/g)
n
108 ± 6 111 ± 7
10.0 ± 0.5 7.4 ± 0.9
142 ± 17 150 ± 24
108 ± 5 137 ± 33a
6 6
Note. Data were obtained after 90 min of reperfusion. Values shown represent the mean ± SE for each group. p values were obtained by t test. a p < 0.001 vs control. DP, developed pressure; CF, coronary flow; CK, creatine kinase concentration; LDH, lactate dehydrogenase concentration in the perfusate; UWBDM, University of Wisconsin solution modified with 2,3-butanedione monoxime and 1 mM Ca2+; n 4 number of hearts in each group.
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hearts preserved for 2, 4, 12, 24, and 24 h (no BDM), respectively. All hearts gained from 47 to 64% weight after 120 min reperfusion, and there were no significant differences between groups. Furthermore, the WG was not related directly to preservation time. Control hearts gained 57 ± 7%, while those preserved for 12 h gained 46 ± 3%, and those for 24 h gained 49 ± 6%. The effects of preservation time on enzyme release (CK and LDH) are shown in Fig. 1. The release of enzymes appeared to show a biphasic effect dependent upon preservation time, which was similar to the effect of preservation time on CO. In hearts preserved for 2, 4, or 12 h the amount of CK or LDH released from the heart was not significantly different from the amount released from fresh hearts during 120 min of reperfusion. However, after 24-h preservation there was a sharp increase in the amount of CK released and a smaller increase in LDH release compared with hearts preserved for 12 h or less. The amounts of CK and LDH in the perfusate from 24-h preserved hearts were significantly greater than those released by fresh hearts.
However, the amounts of enzymes (CK and LDH) in the perfusate from hearts preserved for 2, 4, or 12 h were not significantly greater than the amounts in the perfusate from a freshly perfused heart. Hearts preserved for 24 h without BDM showed LDH and CK release similar to that of hearts preserved for 24 h with BDM, and the differences (±BDM) were not significant at p > 0.05. The results in Table 4 show how preservation with or without BDM for 24 h affected high energy phosphate metabolites in the heart after cold storage. In hearts preserved with BDM, creatine phosphate content was unmeasurable. However, the ATP content was similar to fresh hearts (not significantly different) as was the total adenine nucleotide content. Because the ATP content after 24-h storage with BDM was similar to control hearts we did not measure the concentrations of high energy phosphate after shorter periods of preservation. We presumed that, after shorter periods of preservation the high energy phosphate content would not be significantly less than that observed after 24-h preservation.
FIG. 1. Creatine kinase (CK) and lactate dehydrogenase (LDH) concentration in perfusate after 120 min or reperfusion. Values reflect the mean ± standard error of the mean for each group. *p < 0.05 vs control; # P < 0.001 vs control.
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COLD STORAGE OF THE HEART TABLE 4 Myocardial High Energy Phosphate Content
Control 24 UWBDM 24 UW
Cr. Ph
AMP
ADP
ATP
TAN
n
6 ± 5.1 0 0
3.8 ± 0.44 4.6 ± 0.23 11.9 ± 3.8a,b
9.8 ± 0.49 11.2 ± 1.1 10.4 ± 2.6
37.0 ± 2.0 33.5 ± 1.3 12.4 ± 3.6a,b
50.0 ± 2.4 49.3 ± 1.8 34.6 ± 7.4a,b
10 12 8
Note. High energy phosphates were assayed by high performance liquid chromatography after cold storage. Values are nm/mg of protein and represent the mean ± standard error for each group. P values were obtained by ANOVA + Tukey–Kramer post test. Cr. Ph., creatine phosphate; AMP, adenosine monophosphate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; TAN, total adenine nucleotides; n, number of hearts in a group. a p < 0.05 vs control. b p < 0.05 vs 24 UWBDM.
Hearts preserved without BDM for 24 h showed a loss of high energy phosphates compared with fresh hearts or those preserved for 24 h with BDM. ATP decreased by 68% compared with control hearts and was significantly less than in hearts preserved for 24 h with BDM. DISCUSSION
The working heart is considered to be a more rigorous test of cardiac function than the Langendorff nonworking heart because the heart pumps perfusate against a hydrostatic pressure rather than against a fluid-filled latex balloon placed in the left ventricle. This may resemble more closely the work that the heart would be required to do after transplantation and represent more closely the work required to maintain the circulation of the recipient. Galinanes and Hearse (2) compared the effects of global ischemia on the recovery of function of rat hearts reperfused by either the Langendorff or working model. They showed that the working model was more rigorous. Although the recovery of function was similar after 30 min of warm ischemia, after 45 min of global ischemia the recovery of function was considerably less in the working model (4%) than in the Langendorff model (34%). Thus, for short periods of injury (such as short periods of preservation or global warm ischemia) either model may be appropriate to measure recovery of cardiac function. For longer periods of injury the working model may be a better test of how well the heart recovers function and may be more relevant to long term
heart preservation studies to assess the functional competence of the heart. This conclusion appears to be borne out by the results presented here. We show that the recovery of cardiac function (LVDP) as measured in the Langendorff model after 24-h preservation with UWBDM was nearly 100% (similar to a previous study) but not nearly so good when measured in the working model. After 24-h preservation with UWBDM, recovery of cardiac function (CO) was poor when tested in the working model and equal to only 36% of fresh hearts. From our results we postulate that there are two phases of injury to the heart due to preservation. The first phase is reversible injury, and the second phase is irreversible injury. The initial injury occurs rapidly after flushout with a hyperkalemic, hypothermic preservative such as the UW solution. After only 2 h of cold storage CO is reduced by about 22% of controls. After 4 or 12 h of storage there is no significantly greater decrease in CO compared with the decrease at 2 h. During this initial phase of injury significant increases in cellular damage and increased leakage of intracellular enzymes did not occur. The concentrations of both CK and LDH released into the reperfusion fluid were similar at 2, 4, and 12 h and also similar to fresh hearts. At 24-h preservation, however, there was a significant decrease in CO to 36% of fresh hearts. The CO at 24 h was significantly different than at 2, 4, or 12 h. Also, after 24-h preservation there was a large increase in the amount of CK and LDH released into the reper-
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fusion medium, indicative of cell membrane damage. The increased degree of injury at 24 h does not appear due to a loss of ATP, which is near normal after 24-h storage. There is evidence in the literature which is somewhat supportive of the biphasic nature of injury to the preserved heart. For instance, it is well known that hearts tolerate about 4 h of cold storage, and this is currently the accepted limits for human heart preservation. Recently, clinical heart preservation has been extended in a few cases to 8 h (1). Also, Jeevanandam et al. (5) have shown in the orthotopic heart transplant model using the baboon that hearts preserved for about 12 h in UW solution are fully viable. However, hearts preserved for longer periods were irreversibly injured, and no animal survived after transplantation. The initial injury that occurs with cold flushout of the heart does not appear, therefore, to lead to failure of the transplant. However, it is well known that up to 25% of the preserved hearts transplanted within about 4 h develop serious complications related to preservation/ reperfusion injury (3). Thus, even though the injury may be reversible, it is serious, and methods to suppress this initial injury may be necessary to improve cardiac preservation. Currently, methods to suppress the injury caused by short term preservation involve some form of suppression of reperfusion injury. Drugs such as inotropes, vasodilators, and calcium-regulating agents may be important in hearts preserved for short periods of time. The greater degree of injury occurs in the second phase, after 12-h storage, and what causes this injury and how to suppress it are not known. Hearts preserved for long periods of time may not be amenable to improvement by treating the reperfusion injury with various drugs because the hearts are irreversibly damaged by the long period of cold ischemia. Why hearts do not tolerate preservation greater than about 12 h is not known. Other transplantable organs (liver, kidney, pancreas) are well known to tolerate up to 3 days of preservation in the UW solution. We proposed earlier that one of the limitations to successful long
term heart preservation was ischemic contraction induced by the loss of ATP (14). The loss of ATP in the other transplantable organs is well tolerated, and because of the low concentration of contractile proteins in these organs they do not undergo the type of contracture seen in cardiac muscle. We showed that BDM suppressed ischemic contracture, and we proposed that this agent, or a similar method to suppress ischemic contracture, would be a necessary component of effective long term heart preservation. In this study we show that although CO is depressed after 24-h preservation in UWBDM, without BDM the loss of function after 24-h storage is even greater than with BDM in the UW solution. Thus, BDM may be an important agent in preservatives used for long term heart preservation. However, BDM is not sufficient by itself to extend safe preservation times beyond about 12 h, and other modifications of the preservative or method of preservation will probably be needed to accomplish the goal of truly long term (30–48 h) heart preservation. In summary, this study shows that simple cold storage of the heart with the UWBDM does not appear effective for 24-h preservation when tested in the isolated perfused, working, rabbit heart model. The goal of cardiac preservation is to obtain a time of preservation that will allow effective sharing of hearts on a national level so that no heart is wasted because of lack of sufficient preservation capabilities. For other transplantable organs this period of time is about 24 h. Most livers, kidneys, and pancreases cold stored in UW solution are transplanted within 24 h of harvest, which is sufficient for national sharing. Very few organs are lost because they have been preserved for a period of time that is considered unsafe. Thus, it appears that clinical heart preservation, for optimal utilization of hearts, should be for at least 24 h. To have assurance that the hearts preserved for 24 h would be life sustaining, a demonstration of 30 or more h of successful preservation in the orthotopic transplant model in the laboratory is probably necessary. Current studies indicate that this may be difficult to obtain by simple cold storage. Instead, for successful heart pres-
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ervation the method of choice may be continuous perfusion, which has been shown to give the longest and best quality preservation of the kidney (7), liver (8), and heart (10).
9.
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1. D’Alessandro, A. M., Southard, J. H., Love, R. B., and Belzer, F. O. Organ preservation. Surg. Clin. North Am. 74, 1083–1095, (1994). 2. Galinanes, M., and Hearse, D. J. Assessment of ischemic injury and protective interventions: The Langendorff versus the working rat heart preparation. Can. J. Cardiol. 6, 83–91, (1990). 3. Hauptman, P. J., Aranki, S., Mudge, G. H., Jr., Couper, G. S., and Loh, E. Early cardiac allograft failure after orthotopic heart transplantation. Am. Heart J. 127, 179–186, (1994). 4. Jamieson, N. V., Sundberg, R., Lindell, S., Claesson, K., Moen, J., Vreugdenhil, P. K., Wight, D. G., Southard, J. H., and Belzer, F. O. Preservation of the canine liver for 24–48 hours using simple cold storage with UW solution. Transplantation 46, 517– 522, (1988). 5. Jeevanandam, V., Auteri, J. S., Marboe, C. C., Hsu, D., Sanchez, J. A., and Smith, C. R. Extending the limits of donor heart preservation: A trial with University of Wisconsin solution. Transplant. Proc. 23, 697–698, (1991). 6. Jeevanandam, V., Barr, M. L., Auteri, J. S., Sanchez, J. A., Fong, J., Schenkel, F. A., Michler, R. E., Smith, C. R., and Rose, E. A. University of Wisconsin solution versus crystalloid cardioplegia for human donor heart preservation: A randomized blinded prospective clinical trial. J. Thorac. Cardiovasc. Surg. 103, 194–198, (1992). 7. McAnulty, J. F., Vreugdenhil, P. K., Lindell, S., Southard, J. H., and Belzer, F. O. Successful 7-day perfusion preservation of the canine kidney. Transplant. Proc. 25, 1642–1644, (1993). 8. Pienaar, B. H., Lindell, S. L., Van Gulik, T., Southard, J. H., and Belzer, F. O. Seventy-two-hour preserva-
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