Effects of Butanedione Monoxime and Temperature on Prolonged Cardiac Storage

Effects of Butanedione Monoxime and Temperature on Prolonged Cardiac Storage Ji Zhang, MD, Robert D. Furukawa, BSc, Stephen E. Fremes, MD, Donald A. G. Mickle, MD, and Richard D. Weisel, MD Divisions of Cardiovascular Surgery and Department of Clinical Biochemistry, Centre for Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada

Background. The optimal temperature for cardiac allograft storage remains controversial. We conjectured that supplementation of the potent cardioprotective agent 2,3-butanedione monoxime with calcium may improve allograft storage and make the precise storage temperature less critical. Methods. Hearts were harvested from Sprague-Dawley rats (250 to 350 g), mounted on a Langendorff apparatus, and instrumented with an intraventricular balloon. Hearts were flushed and stored with either unmodified University of Wisconsin solution (UWS) or UWS supplemented with 10 mmol/L of 2,3-butanedione monoxime and calcium 0.1 mmol/L (BDM). Hearts were then subjected to 12 hours of storage at one of five temperatures (0°, 4°, 8°, 12°, or 16°C) in a complete 2 3 5 factorial design (n 5 6/group). Data are reported either as a percentage of the prestorage results or as an absolute value (mean 6 standard deviation). Results. Recovery of developed pressure (p < 0.0001),

coronary flow (p < 0.0001), and diastolic volume (p < 0.001) were significantly enhanced, whereas creatine kinase (p < 0.0001) and lactate dehydrogenase release (p < 0.0001) were reduced in the BDM versus the UWS groups. In both the BDM and UWS storage groups, recovery was better at temperatures of 8°C or less than at 12°C or more. The single preferred temperature was 4°C, significantly better than 0°C with unmodified UWS, while similar to 0° and 8°C with BDM. Adenine nucleotide values were decreased equally in the BDM and UWS hearts, but preservation was enhanced at 0°C compared with all warmer temperatures. Conclusions. We conclude that 4°C is the preferred temperature for prolonged cardiac storage with UWS and that the inclusion of 2,3-butanedione monoxime with calcium 0.1 mmol/L markedly enhances recovery for storage temperatures of 8°C or less. (Ann Thorac Surg 1997;63:388 –94) © 1997 by The Society of Thoracic Surgeons

T

Material and Methods

he addition of 2,3-butanedione monoxime at 30 mmol/L and calcium at 1.0 mmol/L to University of Wisconsin solution (UWS) has been reported by Stringham and associates [1, 2] to significantly enhance cardiac recovery after prolonged hypothermic storage of the rabbit allograft. In similar experiments performed at our institution using rat hearts, we initially demonstrated that the optimal calcium concentration for cardiac storage with UWS was 0.1 mmol/L [3]. In subsequent experiments we identified that the preferred concentrations of 2,3-butanedione monoxime and calcium were 10 mmol/L and 0.1 mmol/L, respectively [4]. The optimal temperature for cardiac allograft storage remains controversial. We conjectured that supplementation of the potent cardioprotective agent 2,3-butanedione monoxime with calcium may improve allograft storage and make the precise storage temperature less critical. The following series of experiments were performed in rodent hearts to answer these questions. Accepted for publication Aug 3, 1996. Presented in part at the 48th Annual Meeting of the Canadian Cardiovascular Society, Toronto, Ont, Canada, Oct 1995. Address reprint requests to Dr Fremes, Division of Cardiovascular Surgery, Sunnybrook Health Science Centre, 2075 Bayview Ave, H405, Toronto, ON M4N 3M5, Canada.

© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

Hearts were obtained from Sprague-Dawley rats (250 to 350 g). All animals received humane care in compliance with “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication No. 85-23, revised in 1985). Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital. Heparin, 200 units, was administered intravenously. A median sternotomy was performed and the hearts rapidly excised and immersed in chilled normal saline. Subsequently, the hearts were perfused after excision in a Langendorff apparatus with filtered Krebs-Heinseleit buffer (composition in mmol/L: NaCl, 118; KCl, 4.7; KH2PO4, 1.2; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25; and glucose, 11) with a pressure of 100 cm H2O. The hearts were ventricularly paced at 300 beats/ min. The reservoirs and conduits were water-jacketed at 37°C. The perfusate was gassed with 95% oxygen and 5% carbon dioxide and the pH adjusted to 7.4. A saline-filled balloon was inserted into the left ventricle through a left atriotomy and fixed in position with 5-0 silk. The balloon volume was varied in 0.01- or 0.02-mL increments from 0 to 0.4 mL, not to exceed an end-diastolic pressure of 30 mm Hg. Data were obtained after a 30-minute stabilization period before storage and after 45 minutes of 0003-4975/97/$17.00 PII S0003-4975(96)00894-6

Ann Thorac Surg 1997;63:388 –94

ZHANG ET AL BUTANEDIONE MONOXIME AND TEMPERATURE

389

The entire coronary effluent was collected. Enzyme release was performed with an Hitachi automatic analyzer 737 (Hitachi Ltd, Tokyo, Japan) and Olympus 800 (Olympus Corp, Lake Success, NY), respectively, using spectrophotometry at 340 nm. The results of these studies are recorded as international units per gram of dried weight. In parallel experiments, levels of adenine nucleotides and nucleosides were assessed by high-performance liquid chromatography as described previously [8]. Measurements were obtained in Langendorff-perfused hearts after the 30-minute stabilization interval or at the end of storage.

Protocol Functional data were obtained after 30 minutes of Langendorff perfusion before ischemia. Hearts were then subjected to 12 hours of storage after aortic root flushing (15 mL/kg) and storage (30 mL) with either unmodified UWS or UWS supplemented with 10 mmol/L 2,3butanedione monoxime and 0.1 mmol/L calcium (BDM). Hearts were stored at one of five temperatures (0°, 4°, 8°, 12°, and 16°C) in a complete 2 3 5 factorial design. There were 6 to 7 animals/group for functional assessment and 6 animals/group for biopsy experiments.

Fig 1. The recovery of developed pressure (mean 6 standard deviation, n 5 6/group) was enhanced in the BDM groups in comparision with unmodified UWS hearts. Zero to 8°C were the preferred storage temperatures in the BDM group and 4°C with unmodified UWS. (BDM 5 University of Wisconsin solution supplemented with 2,3-butanedione monoxime 10 mmol/L and calcium 0.1 mmol/L; UWS 5 University of Wisconsin solution.)

reperfusion after storage. Defibrillation was performed postoperatively as required. All hearts were ventricularly paced during the reperfusion phase at the same heart rate as measured before storage. Developed pressure was recorded before and after storage at a balloon volume associated with an enddiastolic pressure of 20 mm Hg. Diastolic function was initially assessed by evaluating the balloon volume before and after reperfusion at an end-diastolic pressure of 20 mm Hg. Compliance curves were assessed by linear regression analysis of end-diastolic pressure–volume data to calculate the slope and x-intercept as conducted in previous experiments [3–7]. Coronary flow was obtained in duplicate by timed collection in the emptying beating state. Assessment of creatine kinase release and lactate dehydrogenase release was performed during the 45-minute reperfusion phase after the storage interval.

Statistical Analysis Data analysis was facilitated using Statistical Analysis System software (SAS Institute, Cary, NC) and a microcomputer. Variables are expressed as the mean 6 standard deviation of the original value or as a percentage of control. Data analysis was performed with a two-way analysis of variance, and between-group differences were specified with Duncan’s multiple range test or the least squares mean test as appropriate [9]. Diastolic function was assessed with a repeated-measures analysis of variance for evaluation of the slope and x-intercept from before to after storage and additionally as a multivariate analysis of variance simultaneously evaluating the percentage change of slope and x-intercept [9]. Statistical significance is assumed for a p value of less than 0.05.

Table 1. Diastolic Functiona Temperature (°C) 0 4 8 12 16 p Value (analysis of variance)

Slope % Prestorage

X-Intercept % Prestorage

UWS

BDM

UWS

BDM

788.8 6 369.4 323.8 6 324.7 205.8 6 70.6 897.2 6 618.0 1,794.5 6 918.4c Temperature: p , 0.0001

121.0 6 16.3 109.0 6 13.8 122.8 6 10.8 286.5 6 98.7b 766.3 6 397.0b,c Solution: p , 0.0001 Temperature 3 solution: p 5 0.0290

8.8 6 14.6 21.2 6 20.1 10.1 6 44.0 2.0 6 8.4 4.9 6 3.9 Temperature: p , 0.0001

108.6 6 34.1b 103.3 6 12.4b 84.3 6 40.8b 212.5 6 18.9c 1.1 6 8.2c Solution: p , 0.0001 Temperature 3 solution: p , 0.0001

b

a There was a significant increase in the slope of the end-diastolic pressure–volume curves from pre- to poststorage that was limited in the BDM groups. In the UWS groups, the end-diastolic pressure–volume curves were shifted to the left (decreased x-intercept), which was minimized with BDM indicating b p, better preservation of left ventricular compliance. The optimal temperature was 0° to 8°C in the BDM group and 4 to 8°C in the UWS hearts. c p , 0.05 versus 4°C. 0.05 versus UWS.

UWS 5 University of Wisconsin;

BDM 5 UWS supplemented with 2,3-butanedione monoxime 10 mmol/L and calcium 0.1 mmol/L.

390

ZHANG ET AL BUTANEDIONE MONOXIME AND TEMPERATURE

Ann Thorac Surg 1997;63:388 –94

Fig 2. Representative individual left ventricular end-diastolic pressure–volume curves are depicted before storage (Prestorage) and after 12 hours of storage at 4°C (Post-storage). The upper panel was obtained using University of Wisconsin solution supplemented with 10 mmol/L 2,3butanedione monoxime and 0.1 mmol/L calcium (BDM), and the lower panel was acquired using unmodified University of Wisconsin solution (UWS). The left ventricular end-diastolic pressure–volume curve was shifted upward and to the left (increased slope and decreased x-intercept) with unmodified University of Wisconsin solution yet minimally affected with BDM.

Results BDM Versus UWS Poststorage developed pressure, reported as a percentage of the prestorage baseline value, is presented in Figure 1. Developed pressure was reduced after 12 hours of storage compared with prestorage results in both the UWS and BDM groups; however, the decrement in function was significantly less with BDM compared with unmodified UWS at all study temperatures. The diastolic functional parameters are presented in Table 1. Diastolic function was decreased after storage according to both slope and x-intercept criteria (curves shifted upward and to the left), but the deterioration observed for BDM was significantly less than with UWS alone. Representative individual diastolic function curves emphasizing these changes are depicted in Figure 2. Diastolic function was also assessed by evaluating the left ventricular balloon volume both before storage and after reperfusion at an end-diastolic pressure of 20 mm Hg. Preservation of diastolic volume was significantly enhanced in the BDM compared with the UWS group at the 0° to 8°C temperatures (Fig 3). Coronary flows were reduced for both the BDM and

Fig 3. Diastolic volume at a left ventricular end-diastolic pressure of 20 mm Hg is presented as a percentage of the prestorage value (mean 6 standard deviation, n 5 6/group). Recovery of diastolic function was improved with University of Wisconsin solution (UWS) supplemented with 10 mmol/L 2,3-butanedione monoxime and 0.1 mmol/L calcium (BDM) for temperatures 8°C or less. The optimal storage temperature was 0° to 8°C with BDM and 4° to 8°C with unmodified University of Wisconsin solution.

Ann Thorac Surg 1997;63:388 –94

ZHANG ET AL BUTANEDIONE MONOXIME AND TEMPERATURE

Fig 4. Poststorage coronary flow (mean 6 standard deviation, n 5 6/group) was greater with University of Wisconsin solution (UWS) supplemented with 10 mmol/L 2,3-butanedione monoxime and 0.1 mmol/L calcium (BDM) versus unmodified University of Wisconsin solution (UWS) for storage at 0°, 4°, and 8°C. Results for the BDM groups were significantly greater at 0° to 8°C than at 12° to 16°C. There was no significant temperature effect detected in the UWS hearts.

UWS groups relative to their respective prestorage values. The BDM groups showed significantly higher flows than the UWS groups at 0° to 8°C (Fig 4). Creatine kinase and lactate dehydrogenase release results are presented in Table 2. Enzyme release was significantly reduced with BDM at each of the study temperatures except 12°C. The biopsy results are presented in Tables 3 and 4. There was no overall BDM effect for reduction of adenosine triphosphate level and total adenine nucleotide depletion. Values were significantly depressed compared with control results.

Temperature In both the BDM and UWS storage groups, recovery was better at temperatures of 8°C or less than at 12°C or more. In the BDM storage groups, 4°C provided superior protection and 16°C inferior according to developed pres-

391

sure (81.8% 6 6.1% versus 28.6% 6 12.0%) (see Fig 1), coronary flow (93.8% 6 7.2% versus 51.0% 6 9.1%) (see Fig 4), diastolic volume (97.8% 6 5.9% versus 11.0% 6 7.5%) (see Fig 3), and both the slope and the x-intercept of the end-diastolic pressure-volume curves (see Table 1), as well as the cardiac release of creatine kinase and lactate dehydrogenase (see Table 2). There were no significant temperature differences between 0°, 4°, or 8°C for any of the measured end points. Similarly, in the unmodified UWS groups, the best recovery occurred after 4°C storage and the worst recovery with 16°C. This was true for developed pressure (56.5% 6 16.1% versus 7.0% 6 5.0%) (see Fig 1), diastolic volume (35.4% 6 21.1% versus 6.8% 6 2.9%) (see Fig 3), and the slope of the end-diastolic pressure-volume curves (see Table 1), in addition to the cardiac release of creatine kinase and lactate dehydrogenase (see Table 2). There were no significant temperature effects noted with respect to coronary flow or x-intercept. Recovery of both developed pressure and diastolic volume were significantly greater at 4°C than at 0°C, whereas results did not differ between 4°C and 8°C. Adenosine triphosphate depletion was significantly reduced for hearts stored at 0°C. Storage temperatures at 4°C or more did not provide metabolic protection (see Table 3). Accumulation of inosine monophosphate, inosine, hypoxanthine, and xanthine were greater at temperatures of 4°C or more compared with 0°C, whereas adenosine levels were less (see Table 4).

Comment We hypothesized that recovery of hearts after prolonged hypothermic storage would be enhanced with the addition of 2,3-butanedione monoxime and calcium and that the precise storage temperature would be less critical. The UWS was designed with an intracellular concentration of cations to favorably limit ion fluxes during hypothermia (in association with reduced Na-K ATPase activity), which may be inappropriate at warmer temperatures [10]. The calcium inhibitory properties of BDM could extend the effective temperature window of UWS. Improved preservation of developed pressure, coronary flow, diastolic functional parameters, and limitations of

Table 2. Cardiac Enzyme Releasea CK Release (IU/g)

Temperature (°C) 0 4 8 12 16 p value (analysis of variance)

LDH Release (IU/g)

UWS

BDM

UWS

BDM

1,046 6 339.1 859.1 6 316.0 1,142.9 6 802.3 1,540.3 6 445.9 3,166.3 6 853.1c Temperature: p , 0.0001

144.2 6 79.6b 144.9 6 69.5b 436.3 6 204.7b 1,439.9 6 323.7c 2,024.1 6 592.2b,c Solution: p , 0.0001

251.9 6 114.7 232.4 6 90.5 307.5 6 227.8 428.8 6 116.6 817.3 6 263.4c Temperature: p , 0.0001

38.1 6 14.8b 37.7 6 15.8b 112.7 6 55.7b 383.1 6 84.6c 549.1 6 155.6b,c Solution: p , 0.0001

a Cardiac release of both creatine kinase (CK) and lactate dehydrogenase (LDH) were decreased in the BDM versus UWS hearts. Cardiac enzyme release b c p , 0.05 versus UWS; p , 0.05 versus 4°C. increased progressively for temperatures 8°C or more.

Abbreviations are as in Table 1.

392

ZHANG ET AL BUTANEDIONE MONOXIME AND TEMPERATURE

Ann Thorac Surg 1997;63:388 –94

Table 3. Adenine Nucleotide Resultsa Adenine Nucleotide (mmol/g) ATP ADP AMP TAN CP

Temperature (°C) Group

0

4

8

12

16

BDM UWS BDM UWS BDM UWS BDM UWS BDM UWS

5.62 6 2.72 4.09 6 1.58 5.92 6 0.91 6.04 6 1.89 2.50 6 1.13 3.55 6 1.23 14.04 6 2.33 13.68 6 2.28 1.37 6 0.27 1.28 6 0.10

0.74 6 0.22b,c 0.48 6 0.11b 2.94 6 0.74b,c 1.73 6 0.28b 4.11 6 1.87 5.66 6 1.85 7.79 6 1.98b 7.86 6 2.02b 1.76 6 0.97 1.12 6 0.09

0.32 6 0.01b 0.37 6 0.07b 1.63 6 0.12b 1.57 6 0.19b 3.49 6 0.56c 2.68 6 0.17b 5.44 6 0.55b,c 4.62 6 0.14b 1.02 6 0.23 1.67 6 1.35

0.31 6 0.01b,c 0.26 6 0.01b 1.51 6 0.17b 1.45 6 0.05b 4.46 6 0.49b 3.55 6 1.01 6.28 6 0.55b 5.26 6 0.99b 0.91 6 0.23 1.01 6 0.15

0.24 6 0.04b 0.24 6 0.04b 1.32 6 0.09b 1.35 6 0.09b 4.30 6 0.52b,c 3.06 6 0.60 5.86 6 0.58b,c 4.65 6 0.59b 0.97 6 0.02c 0.81 6 0.09

Control (Prestorage) 14.16 6 0.64 4.71 6 0.83 0.41 6 0.16 19.28 6 0.88 11.15 6 3.48

a Levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), total adenine nucleotides (TAN), and b c p , 0.05 versus 0°C. p , 0.05 versus UWS. creatine phosphate (CP) are given as micromoles per gram. Mean 6 standard deviation, n 5 6/group.

Abbreviations are as in Table 1.

cardiac enzyme release was observed for the BDM groups. Temperature was, however, an important determinant of outcome in both the unmodified UWS and BDM groups, with the 0° to 8°C results exceeding those observed at 12° and 16°C. Experiments conducted with isolated rabbit hearts by Stringham and colleagues [1, 2] strongly support the addition of 2,3-butanedione monoxime (and calcium) to UWS. 2,3-Butanedione monoxime, an organic phosphatase inhibitor, exerts its primary effect by reversibly inhibiting myofilament contraction in cardiac muscle, causing a dose-dependent decrease in the myofibrillar sensitivity to calcium-stimulated contraction [11]. Ca21 binding to troponin C is unaffected [12]. Other monitored Ca21 effects include reduced transients according to intracellular fluorescent calcium probes [13], although Marijec and colleagues [14] only observed this after sarcoplasmic inhibition with ryanodine. Furthermore, 2,3-butanedione monoxime causes shortening of the action potential due to inhibition of the slow inward current [12]. A key factor in the effectiveness of BDM is its small

molecular weight (101.1) and uncharged lipophillic nature that allows it to move into and out of the intracellular milieu by a simple flush [1]. 2,3-Butanedione monoxime has been shown to be protective against myocardial contracture in rat heart models of anoxia [15], ischemia/ reperfusion [16], and Ca21 paradox [17] and during metabolic inhibition in cultured myocytes [18]. In the current experiments, we compared unmodified UWS with UWS supplemented wth 10 mmol/L 2,3butanedione monoxime and 0.1 mmol/L calcium. Previous dose-ranging studies performed at our institution demonstrated a modest improvement of calcium supplementation to UWS for 8 hours, 0°C rodent heart storage, with an optimal improvement with 0.1 mmol/L. Subsequent dose-ranging studies evaluated 2,3-butanedione monoxime (0, 10, 30 mmol/L) and calcium (0, 0.1, 1.0 mmol/L) supplementation to UWS and determined that 10 and 0.1 mmol/L were the preferred concentrations [4], and that 2,3-butanedione monoxime was of greater importance than calcium. The optimal concentrations of 2,3-butanedione mon-

Table 4. Adenine Nucleotide Degradation Products Resultsa Component (mmol/g) IMP ADO INO HXN XN

Temperature (°C) Group BDM UWS BDM UWS BDM UWS BDM UWS BDM UWS

0 2.23. 6 1.80 2.77 6 2.12 1.71 6 0.46 1.38 6 0.55 2.57 6 0.23 2.73 6 0.29 0.60 6 0.07 0.51 6 0.09 0.05 6 0.04c 0.07 6 0.02

4

8

12

16

6.05 6 1.25b 6.15 6 0.83b 0.85 6 0.44b 0.87 6 0.36b 3.11 6 0.39b 3.14 6 0.20b 0.86 6 0.09 0.76 6 0.11b 0.09 6 0.02b 0.13 6 0.02b

7.96 6 0.86b,c 5.99 6 0.87b 0.86 6 0.24b,c 0.45 6 0.11b 3.03 6 0.12b 3.29 6 0.32b 1.30 6 0.30b 1.15 6 0.17b 0.16 6 0.02b 0.21 6 0.03b

7.99 6 1.58b 6.16 6 1.20b 0.51 6 0.10b,c 0.32 6 0.11b 3.41 6 0.07b 3.48 6 0.10b 2.16 6 0.37b 1.97 6 0.24b 0.27 6 0.02b 0.30 6 0.02b

5.11 6 0.66b 6.29 6 1.62b 0.37 6 0.05b 0.32 6 0.03b 3.86 6 0.16b 3.74 6 0.21b 3.03 6 0.20b 3.18 6 0.30b 0.36 6 0.04b 0.41 6 0.05b

Control (Prestorage) 0.00 0.09 6 0.020 0.36 6 0.21 0.00 0.00

a Levels of inosine monophosphate (IMP), adenosine (ADO), inosine (INO), hypoxanthine (HXN), and xanthine (XN) are given as micromoles per gram. b c p , 0.05 versus 0°C; p , 0.05 versus UWS. Mean 6 standard deviation, n 5 6/group.

Abbreviations are as in Table 1.

Ann Thorac Surg 1997;63:388 –94

oxime and calcium conflict with experimental findings by Stringham and associates [1, 2], which recommended 30 mmol/L and 1.0 mmol/L. The potential reasons for the discrepancy include variations in protocol (initial Langendorff perfusion after cardiac harvest versus immediate storage [1, 2], storage solution (commercially prepared UWS versus freshly made on site [1, 2]), and species (rodent versus rabbit [1, 2]). Of greater importance is that 2,3-butanedione monoxime was a potent cardioprotective additive in both species. Furthermore, BDM supplementation of St. Thomas’ [2], Stanford [2], and KrebsHenseleit [19] solutions has also been effective in storage experiments. Studies conducted by Keon and associates [20 –22] have challenged the notion that myocardial preservation is optimal at profoundly hypothermic temperatures. Initial investigations performed with Tyrode’s solution (Ca21 2.5 mmol/L) using a human right atrial trabeculae model recommended 12° to 20°C versus 0° to 4°C according to resting and developed force measurements [20]. This work was subsequently extended to the evaluation of St. Thomas’ [21] and intracellular solutions including UWS [22]. The investigations with St. Thomas’ solution confirm previous studies from their laboratories showing improved results with 12°C versus 4°C [21]. The intracellular solutions, UWS and EuroCollins, demonstrated improved recovery at 4°C versus 12°C, compatible with both our previous work using human cell cultures [23] and current results with rodent hearts. Previous studies performed at our institution using isolated human cell cultures demonstrated that temperature was an important predictor of cellular viability as assessed by histologic appearance, cell adhesion, and trypan blue exclusion [23]. Temperatures between 0° and 4°C provided better protection during prolonged storage (24 to 72 hours) than at 8°C. Hearse’s group conducted extensive experimental evaluation of prolonged cardiac storage of the rodent heart with St. Thomas’ Hospital solution [24]. They demonstrated good recovery after 1° to 10°C storage and markedly diminished results with temperatures of 12.5°C or more. A second area of concern related to myocardial preservation with UWS is potential impairment of endothelial-dependent relaxation. Cullen and associates [25] reported that bradykinin-induced release of endotheliumderived relaxing factor was maintained in pulmonary endothelial cells incubated with UWS. Mankad and colleagues [26] stressed that loss of 5-hydroxytryptamineinduced vasodilatation occurred at temperatures of 15°C or more, but was maintained at 4° to 10°C in the isolated rat heart. In similar experiments at 4°C conducted by Cartier and co-workers [27], conflicting results were obtained in that both endothelial-dependent and independent relaxations were impaired with UWS. Nutt and associates [28] described limited myocardial reflow according to thallium imaging techniques in hearts stored for 24 hours in UWS. In our experiments, recovery of total coronary flow was reduced with unmodified UWS at all measured temperatures. In the BDM groups im-

ZHANG ET AL BUTANEDIONE MONOXIME AND TEMPERATURE

393

proved results were observed with the 0° to 8°C temperatures compared with 12° to 16°C. Our original hypothesis was that the precise storage temperature would be less critical in the presence of a potent cardioprotective agent. Our original hypothesis appears confirmed in as much as good functional recovery was observed at temperatures from 0° to 8°C with 2,3 butanedione monoxime supplementation, whereas a narrower temperature window occurred with unmodified UWS (4° to 8°C). One of the limitations of this investigation was the lack of end-reperfusion adenine nucleotide results, which may have correlated better with the mechanical recovery data than the end-storage biopsies. A second limitation was the use of a buffer-perfused rather than a bloodperfused model. The resistance to ischemia of small mammalian hearts appears greater in the blood-perfused versus crystalloid-perfused hearts [29, 30]. We presume that enhanced recovery would be observed in all storage conditions with blood perfusion and the differences between groups would be minimized, although the basic conclusions regarding BDM and temperature would not change. The most significant short-term toxicity noted from 2,3-butanedione monoxime administration (15 to 30 mg/ kg) in human dose–response studies in the 1950s were temporary neurologic changes without permanent sequelae [31]. Physiologic experiments performed using human atrial and ventricular muscle strips demonstrated that the acute cardiac 2,3-butanedione monoxime effects are reversible after withdrawal of the drug [32]. Although long-term toxicity studies are not available, we speculate that BDM use for clinical cardiac allograft preservation would be without important side effects as there would be little systemic exposure of the recipient, and the cardiac effects are well tolerated. In addition, the greater temperature window may facilitate introduction of BDM into clinical practice. We express our appreciation to Mara-Diana Svikis for preparation of the manuscript and to John Bozinovski, MSc, for assistance with the artwork. We are grateful to Peter Meighoo and the staff of the Department of Clinical Biochemistry at the Toronto Hospital for assistance in the cardiac enzyme determinations. We also acknowledge the Multiple Organ Retrieval and Exchange Program and DuPont Canada Ltd for donation of the University of Wisconsin solution used in this study. This study was supported in part by the Heart & Stroke Foundation of Ontario, grant A2854. Stephen E. Fremes is a Research Scholar of the Heart & Stroke Foundation of Ontario.

References 1. Stringham JO, Paulsen KL, Southard JH, Fields BL, Belzer FO. Improved myocardial ischemic tolerance by contractile inhibition with 2,3-butanedione monoxime and calcium. Ann Thorac Surg 1992;54:852– 60. 2. Stringham JO, Paulsen KL, Southard JH, Mentzer RM, Belzer FO. Prolonging myocardial preservation with a modified University of Wisconsin solution containing 2,3-

394

3. 4.

5. 6. 7.

8. 9.

10. 11. 12.

13.

14.

15.

16. 17.

ZHANG ET AL BUTANEDIONE MONOXIME AND TEMPERATURE

butanedione monoxime and calcium. J Thorac Cardiovasc Surg 1994;107;764 –75. Fremes SE, Zhang J, Furukawa RD, Mickle DAG, Weisel RD. Cardiac storage with UW solution, calcium and magnesium. J Heart Lung Transplant 1995;14:916–25. Fremes SE, Zhang J, Furukawa RD, Mickle DAG, Weisel RD. University of Wisconsin solution and butanedione monoxime. Abstracts of the 15th Annual Meeting of the International Society for Heart and Lung Transplantation, San Francisco, CA, April 4 – 8, 1995. Fremes SE, Guo LR, Furukawa RD, Mickle DAG, Weisel RD. Cardiac storage with UW solution and glucose. Ann Thorac Surg 1994;58:368–73. Fremes SE, Furukawa RD, Zhang J, et al. Cardiac storage with UW solution and a nucleoside transport blocker. Ann Thorac Surg 1995;59:1127–33. Fremes SE, Zhang J, Furukawa RD, Mickle DAG, Weisel RD. Adenosine pretreatment for prolonged cardiac storage: an evaluation with St. Thomas’ and UW solution. J Thorac Cardiovasc Surg 1995;110:293–301. Weisel RD, Mickle DAG, Finkle CD, Tumiati LC, Madonik MM, Ivanov J. Delayed myocardial metabolic recovery after blood cardioplegia. Ann Thorac Surg 1989;48:503–7. Spector P, Goodnight JH, Sall JR, Sarle WS. The GLM procedure. In: Luginbuhl RD, Schlotzhauer SD, Parker JD, eds. SAS/STAT guide for personal computers, version 6. Cary, NC: SAS Institute Inc, 1987;549 – 640. Belzer FO, Southard JH. Principles of solid-organ preservation by cold storage. Transplantation 1988;45:673– 6. Li T, Sperelakis N, Teneick RE, Solaro RJ. Effects of diacyl monoxime on cardiac excitation-contraction coupling. J Pharmacol Exp Ther 1985;232:688–95. Gwathmey JK, Hajjar RJ, Solaro RJ. Contractile deactivation and uncoupling of crossbridges: effects of 2,3-butanedione monoxime on mammalian myocardium. Circulation Res 1991;69:1280–92. Perrault CL, Mulieri LA, Alpert NR, Ransil BJ, Allen PD, Morgan JP. Cellular basis of negative inotropic effect of 2,3-butanedione monoxime in human myocardium. Am J Physiol 1992;263:H503–10. Marijec J, Buljubasic N, Stoew DF, Tumer LA, Kampine JP, Bosnjak ZJ. Opposing effects of diacetyl monoxime on contractility and calcium transients in isolated myocardium. Am J Physiol 1991;260:H1153–9. Nayler WG, Elz JS, Buckley DJ. Dissociation of hypoxiainduced calcium gain and rise in resting tension in isolated rat hearts. Am J Physiol 1988;245(Heart Circ Physiol 23): H678 – 85. Nayler WG, Elz JS, Buckley DJ. The stunned myocardium: effect of electrical and mechanical arrest and osmolarity. Am J Physiol 1988;256(Heart Circ Physiol 23):H60 –9. Daly MS, Elz JS, Nayler WG. Contracture and the calcium paradox of the rat heart. Circulation Res 1987;61:560–9.

Ann Thorac Surg 1997;63:388 –94

18. Ikenouchi H, Zhao L, Barry WH. Effect of 2,3-butanedione monoxime on myocyte resting force during prolonged metabolic inhibition. Am J Physiol 1994;267:H419 –30. 19. Vahl CF, Bonz A, Hagl C, Hagl S. Reversible desensitization of the myocardial contractile apparatus for calcium. Eur J Cardiothorac Surg 1994;8:370– 8. 20. Keon WJ, Hendry PJ, Taichman GC, Mainwood GW. Cardiac transplantation: the ideal myocardial temperature for graft transport. Ann Thorac Surg 1988;46:337– 41. 21. Hendry PJ, Labow RS, Barry YA, Keon WJ. An assessment of crystalloid solutions for donor heart preservation. J Thorac Cardiovasc Surg 1991;101:833– 8. 22. Hendry PJ, Labow RS, Keon WJ. A comparison of intracellular solutions for donor heart preservation. J Thorac Cardiovasc Surg 1993;105:667–73. 23. Fremes SE, Li RK, Weisel RD, Mickle DAG, Tumiati LC. Prolonged hypothermic cardiac storage with University of Wisconsin solution: an assessment using human cell cultures. J Thorac Cardiovasc Surg 1991;102:666–72. 24. Takahashi A, Braimbridge MV, Hearse DJ, Chambers DJ. Long-term preservation of the mammalian myocardium. J Thorac Cardiovasc Surg 1991;102:235– 45. 25. Cullen S, Haworth SG, Warren JD. Donor organ preservation fluids differ in their effect on endothelial cell function. J Heart Lung Transplant 1991;10:999 –1003. 26. Mankad P, Slavik Z, Yacoub M. Endothelial dysfunction caused by University of Wisconsin preservation solution in the rat heart. J Thorac Cardiovasc Surg 1992;104:1618–24. 27. Cartier R, Hollman C, Dagenais F, Buluran J, Pellerin M, Leclerc Y. Effects of University of Wisconsin solution on endothelium-dependent coronary artery relaxation in the rat. Ann Thorac Surg 1993;55:50– 6. 28. Nutt MP, Fields BL, Sebree LA, et al. Assessment of function, perfusion, metabolism, and histology in hearts preserved with University of Wisconsin solution. Circulation 1992;86 (Suppl 2):333– 8. 29. Walters HL III, Digerness SB, Naftel DC, Waggoner JR III, Blackstone EH, Kirklin JW. The response to ischemia in blood perfused vs crystalloid perfused isolated rat heart preparations. J Mol Cell Cardiol 1992;24:1063–77. 30. Qui Y, Hearse DJ. Comparison of ischemic vulnerability and responsiveness to cardioplegic protection in crystalloidperfused versus blood-perfused hearts. J Thorac Cardiovasc Surg 1992;103:960– 8. 31. Jager BV, Stagg GN. Toxicity of diacetyl monoxime and of pyridine-2-aldoxime methiodide in man. John Hopkins Hosp Bull 1058;102:203–11. 32. Schwinger RHG, Bohm M, Koch A, Morano I, Ruegg JC, Erdmann E. Inotropic effect of the cardioprotective agent 2,3-butanedione monoxime in failing and nonfailing human myocardium. J Pharm Exp Ther 1993;2:778– 86.