Cardioplegic effect of University of Wisconsin solution on hypothermic ischemia of rat myocardium assessed by mitochondrial oxidative phosphorylation

Cardioplegic effect of University of Wisconsin solution on hypothermic ischemia of rat myocardium assessed by mitochondrial oxidative phosphorylation The effectiveness of the University of Wisconsin solution and the Collins' M solution for preservation of rat hearts was compared by examining histologic appearance, tissue water content, and mitochondrial respiratory functions after prolonged hypothermic storage and subsequent heterotopic transplantation. Survival of transplanted hearts after 5 days of reperfusion was markedly lowered by storage in Collins' M solution for 15 hours. Hearts stored in University of Wisconsin solution for 10 hours showed no increase in myocardial necrosis after 5 days of reperfusion, whereas hearts stored in University of Wisconsin solution for 15 hours and Collins' M solution for 10 and 15 hours showed a significant increase in tissue necrosis. University of Wisconsin solution reduced tissue swelling during hypothermic storage, whereas Collins' M solution did not cause such reduction. The yield of mitochondrial protein after reperfusion was significantly decreased by storage in either solution, especially after 15 hours in Collins' M solution. Mitochondrial oxidative phosphorylation was significantly inhibited by storage, especially by storage in Collins' M solution and subsequent reperfusion. These results indicate that myocardial injury, after prolonged ischemia and reperfusion, results in a decrease in functionally and structurally intact mitochondria that is dependent on preservation conditions. University of Wisconsin solution protects isolated hearts against ischemia and reperfusion injury possibly by preventing ceUuIar and mitochondrial deterioration. (J THORAC CARDIOVASC SURG 1993;106:502-10)

Hiroshi Yano, MD, Hitoshi Takenaka, PhD,a Toshio Onitsuka, MD, PhD, Yasunori Koga, MD, PhD, and Minoru Hamada, MD, PhD,a Miyazaki, Japan

Cardiac transplantation has been used as an ultimate treatment for severe heart failure. One of the remaining problems concerning cardiac transplantation is limited From the Second Department of Surgery and Department of Hygiene," Miyazaki Medical College, Miyazaki, Japan. This study was supported by a general grant, and Dr. Takenaka was supported in part by a grant-in-aid for scientific research (C) from the Ministry of Education, Science and. Culture of Japan. University of Wisconsin solution and Collins' M solution were donated by DuPont Pharmaceuticals, Japan (Tokyo, Japan) and Midori Juji Co. (Osaka, Japan), respectively. Received for publication Feb. 7, 1992. Accepted for publication Sept. 24, 1992. Address for reprints: Dr. Minoru Hamada, Department of Hygiene, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazakigun, Miyazaki, 889-16, Japan. Copyright

@

1993 by Mosby-Year Book, Inc.

0022-5223/93 $1.00 +.10

502

12/1/43084

availability of donor hearts because of the difficulty associated with long-distance heart procurement. In clinical cases, the acceptable period for heart preservation is, empirically, about 4 hours,' although longer successful preservation has been reported in laboratory animals under well-controlled conditions.i Extension of the limit for viable heart preservation has been expected in human organs. Belzer and Southard.' have developed a useful storage solution known as the University of Wisconsin (UW) cold storage solution, which is an excellent preservative of the human pancreas," liver,5 and kidney." The UW solution has recently been used in experiments with dog," rabbit.! and rat9- 12 hearts in an isolated working model and in a heterotopic transplant model after longterm preservation. The UW solution has been shown to minimize hypothermia-induced tissue swelling, intracellular acidosis, deterioration in histologic appearances, and reperfusion injury while restoring high-energy phosphate

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 3

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_ .

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Fig. 1. Experimental protocol. Hearts were arrested with a hyperkalemic solution and flushed with preservation solution at 4° C before hypothermic storage. After 10 or 15 hours of storage, hearts were heterotopically transplanted. Reperfusion of donor hearts was started by declamping aorta 30 minutes after start of anastomosis. Tissue water content and yields and functions of mitochondria of hearts in nonreperfused, I-day reperfused, and 5-day reperfused groups were determined. Size of necrosis was assessed in 5-day reperfused hearts. levels, electrolyte content, and cardiac function. However, the basic mechanisms that are responsible for the solution's effectiveness are not fully understood. We designed this study to compare cardioplegic effectiveness of the UW solution with that of the Collins' M (CM) solution, which resembles the UW solution in the electrolyte composition and the total osmotic pressure, by examining mitochondrial respiratory functions and myocardial necrosis after prolonged reperfusion in vivo in heterotopically transplanted hearts. Our results indicate that the UW solution, when compared with the CM solution, is more effective in reducing the development of necrosis and in maintaining mitochondrial oxidative phosphorylation. Cellular integrity, as assessed by yield of mitochondrial protein, was less disturbed after storage in the UW solution than it was in the CM solution.

Materials and methods Animals. Our study followed the Guide for Animal Experimentation issued by Miyazaki Medical College, which was based on the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). Specific pathogen-free, inbred Lewis male rats were kept under specific pathogen-free conditions for 2 weeks in polycarbonate cages with stainless steel drain boards at the Experimental Animal Center of Miyazaki Medical College and were fed a conventional commercial diet (CE-12; Clea Japan Inc., Tokyo). Rats were randomly assigned as donors or recipients of hearts. Preparation of donor hearts. Rats (10 weeks of age, 235 to 265 gm) were anesthetized with pentobarbital sodium (40 mg/kg) intraperitoneally. After intravenous heparinization (250 IV) of donor rats, the superior and inferior venae cavae

were clamped immediately after thoracotomy. Blood in the cardiac chamber was slowly flushed with 5 ml of cold saline solution from the inferior vena cava through the aorta with care and to avoid applying excess pressure on the right atrium. Three minutes after thoracotomy, hearts were arrested by infusing 3 ml of a hyperkalernic solution containing potassium citrate (24.7 mmol/L), NaCI (70.9 mmol/L), and MgS04 (100 mmol/L) (pH 7.4) at 4° C from the aortic arch. While the heart was cooled by cold saline solution in the thoracic cavity, an occlusive cannula was inserted into the ascending aorta, and 5 ml of the VW or the CM preservation solution, containing 1000 IV/L heparin, was manually perfused. The VW solution was donated by DuPont Pharmaceuticals (Tokyo, Japan) and was composed of potassium lactobionate (100 mmol/L), raffinose (30 mmol/L), KH2P04 (25 mmoljL), MgS04 (5 mmoljL), adenosine (5 mmol/L), reduced glutathione (3 mmol/L), insulin (40 V /L), dexamethasone (16 mg/L), allopurinol (I mmol/L), and hydroxyethyl starch (50 gm/L) (320 mOsrrr/L). The CM solution was donated by Midori Juji Co. (Osaka, Japan) and was composed of NaHC03 (10 mmol/L), KCl (15 mmol/L), MgS04 (6 mmoljL), KH2P04 (15 mmol/L), K2HP04 (85 mmol/L), and glucose (144 mmol/L) (310 mOsm/L). The superior and inferior venae cavae were ligated, the aorta and pulmonary trunk were transected, and the pulmonary veins were ligated while soaking in 4° C saline solution. Fifteen minutes after the onset of infusion of hyperkalemic solution, hearts were removed and stored in 200 ml of the preservation solution at 4°C. Hypothermic preservation. Hearts were randomly assigned to four storage groups (Fig. 1) according to the type of preservation solution (UW or CM solution) and the storage period (10 or 15 hours). Control group hearts were prepared by immersion in cold saline solution for 15 minutes, the time between potassium arrest and immersion, without storage in the preservation solution. Heart transplantation. Heterotopic heart transplantation'! was carried out while hearts were topically cooled by cold saline solution. In brief, an end-to-side anastomosis was performed with an 8-0 polypropylene suture (Ethicon, Inc., Somerville,

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Table I. Survival of grafts after 1 and 5 days of reperfusion After 5 days

After 1 day Reperfusion group

Control UW-IO CM-IO UW-15 CM-15

Surviving grafts/ total grafts

%

6/6 6/6 6/6 6/6 6/9

!OO 100 100 !OO 67

Surviving grafts/ total grafts

%

10/10 11/11 !0/1I 11/13 9/14

100 !OO 91 85 64

Graft survival was evaluated by palpation of transplanted hearts after storage in UW solution or eM solution for 10 or 15 hours.

N.J.) between the donor's aorta and the recipient's aorta and between the donor's pulmonary trunk and the recipient's vena cava; the procedure was completed within 25 minutes. Reperfusion of the donor heart was started by declamping the aorta 30 minutesafter beginningthe anastomosis. Recipientrats were given free access to food and water, and pulses of the transplanted hearts were examined daily by palpation on the recipient's abdomen to evaluate survivalof the transplanted hearts. After 1 or 5 days of reperfusion (Fig. 1), working heart grafts were arrested by infusionof 3 ml of the ice-coldhyperkalemic solution from the aortic root immediately after clamping the recipient's aorta at both sidesof the anastomotic portions.The hearts were excisedand examinedfor the development of tissue necrosis. The water content of the tissue was measured, and mitochondria were isolated. Evaluation of myocardial necrosis. Hearts were arrested in the diastolicstage,excised,and immediatelyrinsedincoldsaline solution. A transverse slice (about 2 mm thick) was obtained from a ventricle by cutting in parallel to the atrioventricular sulcus at the midportion between the apex and the base of the heart. The slice was incubated for 10 minutes in 1% triphenyltetrazolium chloride (TIC) and sodium phosphate buffer (lOOmmol/L) (pH 7.4) and fixedin 10%(vol/vol) neutralized formaldehydefor 24 hours in the dark-a modified method of Fishbein and associates.!" Both sides of the slice were photographed at a magnificationof 12.Color imagesof the slicewere stored in an optical magnetic disk recorder and examined on a color image display with the use of a color image processor (SPICCA II; Nippon Avionics, Co., Ltd., Tokyo,Japan), which distinguished the TTC-stained area from the unstained area. The averagedextent of necrosisfrom both sidesof the sliceswas evaluated as the percentage of the unstained area compared with the total cross-sectional area. Estimation of tissue water content. About 100 mg of the apical portion of the hearts was dried for 24 hours at 800 C and at ambient pressure. Relative water content was calculated as (Wet weight - Dry weightr/Wet weight. Isolation of mitochondria. Mitochondria wereisolatedat 4 0 C by the method of Chance and Hagihara 15 with a slight modification.Finelydicedventricularmusclewas homogenized with a Potter-Elvehjem Teflon homogenizer(5 strokes, 500 rpm) in about 15 vol/wt of sucrose (0.25 rnol/L), ethylenediaminetetraacetic acid (I mmol/L), and N-2-hydroxyethylpiperazineN' -2-ethanesulfonate (HEPES, 20 mmol/L) buffer (pH 7.4) in the presenceof N agarse enzyme (5 tug] gm wet tissue).After incubation on ice for 10 minutes, the homogenate was centri-

fugedat 600 g for 10minutesand the resultant supernatant was centrifuged at 8000 g for 20 minutes. The pelletobtained after another centrifugation of the above supernatant at 8000 g for 20 minutes was suspended in 0.3 ml of sucrose (0.25 mol/L), KCI (20 mmol/L), HEPES (20 mmol/L; pH 7.4). Mitochondrial functions were examined immediately after isolation. Mitochondrial respiratory function. Respiratory function of mitochondria in the presenceof succinate as a substrate was determined by measuring oxygenconsumptionwith an oxygen probe (Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio) at 250 C. The reaction was initiated by the additionof 50 ~l of mitochondrialpreparation, 50 ~l of succinate (100 mmol/ L; pH 7.4), and then 5 ~lofadenosinediphosphate (ADP;48.2 mmol/L; pH 7.4) to 1.8 ml of a medium containing sucrose (0.25 mol/L), KCI (20 mmol/L), MgCh (5 mmol/L), potassium phosphate buffer (10 mmol/L; pH 7.4), and 20 mmol/L HEPES (pH 7.4), which had been equilibrated with air by bubbling for at least 20 minutes at 250 C. The initial oxygen content in the medium was assumed to be 450 nanoatom/ml, Respiratory controlindex wascalculated by dividing the rate of oxygenconsumptionafter the additionof ADP (state 3) by the rate beforethe addition of ADP (state 4). Phosphate-to-oxygen ratio (P /0) was calculated by dividing the amount of added ADP by the amount of oxygenconsumed in state 3. Protein concentration in mitochondrial preparations was determined by the method of Lowry and associates.l? with the use of bovine serum albumin as a standard. Concentration of ADP was determined by measuringabsorbanceat 259 nm and by using 15.4 as the millimolar extinction coefficient. Ethylenediaminetetraaceticacid was purchased from Dojindo Laboratories (Kumamoto, Japan), and ADP was purchased from Kohjin Co. Ltd. (Tokyo, Japan). All other reagents used were of analytic grade and purchased from either Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan). Statistical treatment. All values are expressed as mean ± standard deviation. Significant differences among the five groups (control and four storage groups) were assessed with analysisof variance.This analysiswasalso appliedto assess significant differences among groups with l-day, 5-day, and no reperfusion. Significant differences in mitochondrial functions betweenthe nonreperfusedgroupand the reperfusedgroupwere analyzed by Student's unpaired t test. The differencewas considered significant when p < 0.05. To simplify the tables and figures, we-have indicated significantdifferencebetweencontrol and storage groups, between UW and CM groups of the same storage periods,between 10-and 15-hourstoragegroupsin each solution, and between nonreperfused hearts and reperfused hearts.

Results Graft survival. Survival of the graft after I-day reperfusion was as low as 67% in the CM group with IS hours of storage (CM-15). The other groups showed higher survival than did the CM-IS group (Table I). The survival after reperfusion for 5 days remained 100% for the control group and the UW group with 10 hours of storage (UW -10), whereas the remaining groups showed lower survivals. Only beating hearts underwent subsequent assay because nonbeating hearts showed total necrosis.

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 3

Evaluation of myocardial necrosis. Myocardial necrosisafter reperfusion for 5 days is shown in Table II. The extent of necrosis was about 10% in control hearts, which was likely attributable to injury occurring during immersion in cold saline solution for 15 minutes and the subsequent transplantation procedure. There was no significant difference in the necrotic area between control and UW-IO groups. All UW-15 and CM-15 groups and CM groups with 10 minutes of storage (CM-10) showed a significant increase in extent of necrosis compared with the control group, with the CM-15 group demonstrating a significantly larger area of necrosis than the UW-I5 group. Tissue water content. Tissue water content was 76.0% ± 0.4% in immediately excised hearts (n = 5) and 76.4% ± 0.7% in potassium-arrested hearts (n = 5). The controlgroup showed an apparent increase in water content to 79.4% ± 0.6%, indicating that hearts were swollen by 15 minutes of immersion in saline solution (Table III). Tissue water content of hearts after storage in UW solutionwas similar to that of hearts without storage and was significantly lower than that of control hearts, whereas water content in CM groups was significantly higherthan that in the UW groups. No significant difference was observed in either l-day or 5-day reperfusion groups.Tissue water content in the UW-15 group hearts wassignificantlyincreased after reperfusion for 1day, but further reperfusion did not affect the water content. Yet, the water content in the CM-lO group after 5 days of reperfusion was significantly lower than that before reperfusion and after 1 day of reperfusion. All storage groups thus had increased water content after 1 day of reperfusion and decreased water content after 5 days of reperfusion. The water content in hearts after 5 days of reperfusion was still higher than that in immediately excised hearts. Yield of mitochondrial protein. Yields of mitochondria were estimated as yields of protein in the mitochondrial preparations (Table IV). Yields of mitochondrial protein before reperfusion were apparently lowered by storage, although no statistically significant difference among groups was found. Reperfusion for 1 day significantlylowered the yield of mitochondrial protein, and all storagegroups showed a significantly lower yield than did thecontrol group. Further decrement was insignificant in all groups after reperfusion for the following 4 days. No significant difference was found between UW-IO and CM-lO groups after reperfusion for 1 and 5 days. The CM-15 group, however, showed a significantly lowered yield compared with that of the UW-15 group after both 1 and 5 days of reperfusion. These results indicate that changes in heart tissue affecting yields of mitochondrial

Yano et al.

50 5

Table II. Percentage of necrotic area in hearts after 5 days of reperfusion Reperfusion group Control

UW-IO CM-IO UW-15 CM-15

Relative necrotic area (%) 9.87 17.93 24.32 21.25 35.24

± ± ± ± ±

2.85 5.87 8.17** 3.29* 6.28***t

No. of samples

6 6 6 6 5

Relative necrotic area = (TIC - Unstained areal/Total cross-sectional area. Values are the mean ± standard deviation. Significant differences are shown between control group and each storage group (*l and between UW-15 and CM-IS (t).

*tp < O.OS. "p <0.01.

"'p <0.001.

protein occurred during day 1 of reperfusion, regardless of the solution used, and that the change was significant between hearts stored in UW and CM solution for 15 hours. Mitochondrial function. Respiratory functions of mitochondria isolated from control and stored hearts with and without subsequent reperfusion were examined with the use of succinate as a substrate (Fig. 2). State 3 respiration (Fig. 2, A) was significantly decelerated by storage for 15 hours in both UW and CM solutions, although no significant difference was found after 10 hours of storage. Reperfusion caused significantly depressed state 3 respiration compared with that of nonreperfused groups in all cases. In reperfused hearts, the CM-15 group showed a significantly lower rate than did reperfused control hearts. State 4 respiration was apparently but not significantly affected by storage (Fig. 2, B). No significant difference in the rate was observed in control and CM-IS groups between nonreperfused and reperfused hearts, whereas in other storage groups reperfusion significantly decelerated state 4 respiration. No statistically significant difference was found between reperfused hearts. All groups except the OW-I 0 group had their respective respiratory control indexes lowered by storage, compared with the control group (Fig. 2, C). Reperfusion significantly lowered the respiratory control index of nonreperfused hearts in control and CM groups, although there was no significant decrease in UW groups. In reperfused hearts, the CM-15 group showed significantly lower respiratory control indexes than with those ofthe UW-15 group, whereas the other groups were not significantly different. The P /0 ratio of mitochondria isolated from control and UW group hearts were not significantly affected by storage, but both CM groups showed significantly lower P /0 values than did the control group (Fig. 2, D). The P /0 value of CM-IS was much lower than that of

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Table III. Percentage of tissue water content Before reperfusion Reperfusion group Control UW-IO CM-1O UW-15 CM-15

Mean ± standard deviation

No. of samples

± ± ± ± ±

6 7 9 9 7

79.4 76.1 78.5 76.9 79.2

0.6 1.9** l.3t 104* 0.9t

After 5 days

After 1 day 79.1 78.5 79.2 79.2 79.9

± ± ± ± ±

2.2 1.9

1.111 1.0§§ 1.5

(6) (6) (6) (6) (6)

77.8 ± 0.6 7704 ± 0.5 76.6 ± 1.2§ 78.0 ± 0.9 78.4 ± 0.6

(4) (5) (4) (5) (4)

Significant differences are shown between control group and each storage group (*), between UW-IO and CM-I 0 (t), between UW-IS and CM-IS (:j:), between nonreperfused group and reperfused group (§), and between I-day reperfused group and S-day reperfused group
**§§ P < om.

Table IV. Yield of mitochondrial protein Reperfusion group Control UW-IO CM-IO UW-15 CM-15

Before reperfusion 79.2 68.6 68.6 68.1 68.9

± 2.8 ± 4.9 ± 8.0 ± 7.2 ± 7.3

After 1 day 69.7 55.2 51.8 48.1 38.7

± 5.3§§ ± 4.1***§§§ ± 2.0***§§§ ± 3.0***§§§ ± 7A***tt§§§

After 5 days 69.7 54.0 51.3 46.1 35.0

± 2.9§ ± 4.1**§§§ ± 4.1**§§§ ± 6.6***§§§ ± 4.9***ttt§§§

Values are expressed in milligram protein per gram dry weight and the mean ± standard deviation. Numbers of samples are same as those shown in Table III. Significant differences are shown between control group and each storage group (*), between UW-IS and CM-IS (t), between CM-IO and CM-IS (:j:), and between nonreperfused group and reperfused group (§).

t§ p < 0.05,

**:j::j:§§ p < om, ***§§§ p < 0.001.

UW-I5. No significant difference was observed between nonreperfused and reperfused hearts in all fivegroups. In reperfused hearts, no significant difference was found between the control and UW-10 group, but UW-15 and both CM group hearts showed significantly lower P /0 values than those of the control group. Both CM-10 and CM-15 group hearts showed significantly lower P/Os after I-day reperfusion than UW-1O and UW-I5 group hearts, respectively. Oxidative phosphorylation rate, which was calculated as the product of state 3 respiration rate and P /0 value, is shown in Fig. 2, E. In both nonreperfused and reperfused hearts, the oxidative phosphorylation rate of the UW-10 group was not significantly different from that of the control group, but all other stored groups showed significantly decreased rates compared with the control group. In reperfused hearts, storage in CM solution showed a significantly lower oxidative phosphorylation rate than did UW solution in corresponding storage periods. Differences between nonreperfused and reperfused hearts were significant in g~oups other than CM-15. Taking into account the yield of mitochondrial protein (Table IV), the rate of adenosine triphosphate (ATP) synthesis (micromoles per minute per gram of dry tissue), which was calculated from the oxidative phosphorylation rates, could be decreased by reperfusion from 45.2 to 29.4

in the control group, 34.8 to 18.4 in the UW-IO group, 2I.2t08.5 in theCM-1O group, 24.7 to 11.1in the UW-I5 group, and 13.2 to 3.8 in the CM-I5 group. Discussion Transplanted hearts are expected to fully support the circulatory system of recipients immediately after transplantation and prolonged reperfusion. Cardiac performance after transplantation has mostly been predicted by experiments with Langendorff perfusion'Y" and the reperfusion model of the working heart mode. 19,20 The purpose of the present study is to examine the effectiveness of two established organ-storage solutions (UW and CM solutions) for rat myocardium after prolonged hypothermic ischemia and subsequent blood reperfusion in vivo by comparing histologic and biochemical consequences. Irreversible injury sustained by the myocardium was evaluated as necrotic area, tissue edema was evaluated as the water content, and energy-producing capability was evaluated as mitochondrial respiratory functions. Our model of heterotopic transplantation of rat hearts has the advantage that reperfusion with blood in vivo is closer to physiologic circumstances than is a crystalloid perfusion system. Use of animals from a closed colony eliminates the possible contribution of immunologic response to cardiac functions. However, a disadvantage

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 3

YaM et al. 5 0 7

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Fig. 2. Comparison of mitochondrial respiratory functions between nonreperfused (open columns) and reperfused (filled columns) hearts. A, State 3 respiration rates (nanoatom oxygen per milligram protein per minute); B, state 4 respiration rates (nanoatom oxygen per milligram protein per minute); C, respiratory control indexes (ReI); D, P /0; and E, oxidative phosphorylation rate (OPR, nanomole ATP per milligram protein per minute). Number of samples before and after reperfusion is, respectively, 6 and 6 for control, 7 and 6 for UW-IO, 9 and 6 for CM-IO, 9 and 6 for UW-15, and 7 and 6 for CM-15. Bars indicate standard deviation. Significant differences are shown between control group and each storage group (*), between UW-IO and CM-1O or UW-15 and CM-15 (t), between UW-IO and UW-15 or CM-1O and CM-15 (*), and between nonreperfused and reperfused hearts (§). *t*§p < 0.05; **ttH§§p < 0.01; ***ttt§§§p < 0.001.

may be that the absence of reperfused blood in the left ventricular chamber keeps left ventricular muscles unloaded, even though coronary circulation and collateral flow were likely present. This circumstance lead to the

underestimation of myocardial injury derived from changes in hemodynamic function, thus minimizing the evaluation of necrotic area. In spite of the disadvantage, the present model is useful to analyze myocardial injury

508

Yanoetal.

that may occur at low load under physiologic conditions. The TIC method is a superb histologic technique for the evaluation of the necrotic region, 14 but planimetric observation may overlook patchy infarcts in normal myocardium. We therefore used computerized estimation with a color image analyzer in our study to account for patchy infarcts. We applied the TTC method to analyze the size of myocardial necrosis after 5 days of reperfusion, at which time irreversible injury would be established but no fibrosis should have developed." Our study showed that graft survival was decreased and necrotic size was enlarged in a manner dependent on ischemic periods, even though hearts were kept under hypothermic conditions. Hearts stored in the CM solution had worse survivals than did those stored in the UW solution. Necrotic size in the control hearts was as high as 10%. This apparent increment was likely due to myocardial injury occurring during immersion in cold saline solution for 15 minutes (the time between cardiac arrest and start of storage) and the subsequent transplantation procedure. The UW solution significantly reduced tissue necrosis compared with CM solution, especially after 15 hours of storage. This fact is consistent with the findings of Makowka and associates, II who showed that histologic appearance in heterotopically transplanted hearts on the third day after storage in UW solution for 12 to 18 hours was better than those after storage in the Stanford cardioplegic solution. Our finding also agrees well with the report of Yeh and associates,'? which states that hypothermic ischemia for 6 hours in UW solution appeared to be better than that in St. Thomas' Hospital cardioplegic solution in terms of histologic appearance after 1 hour of reperfusion in the working heart mode. These reports indicate that UW solution is capable of preserving hearts in good condition at least in histologic appearance. It should be noted that graft survival and necrotic size were not necessarily correlated. Control hearts showed full survival with 10% necrosis, and CM-l 0 and UW-15 showed the opposite relation between survival and necrotic size. This observation may be explained if survival depends on location of the infarct rather than on the size of the infarct.F though the present study evaluated only infarct size. Future study is expected to analyze both aspects of size and location of myocardial necrosis to understand how necrosis correlates to graft survival. One explanation for the effectiveness of UW solution in protecting the myocardium from ischemia and reperfusion injury has been its high colloidal osmotic pressure, which prevents or reduces tissue edema during hypothermic storage.' DiBona and Powell-' showed the strong correlation between cell swelling and eventual necrosis in

The Journal of Thoracic and Cardiovascular Surgery September 1993

ischemic dog myocardium. In the present study, tissue water content in hearts immediately after arrest with the hyperkalemic solution was similar to that in immediately excised hearts, indicating that increased tissue water content in control hearts was due to tissue swelling during immersion in cold saline solution for 15 minutes. Taking into consideration that the tissue water content of the myocardium before hypothermic storage was about 76%, it is logical to assume that the UW solution protected the myocardium from swelling, whereas the control and CM groups had increased tissue water content. This effect may be attributed to the inclusion of impermeants in UW solution. Apparently, increased water content after 1 day of reperfusion was similarly decreased after 5 days of reperfusion in stored groups. However, the values were still higher than the water content in beating hearts, indicating that equilibrium between heart tissue and plasma fluid had not been reached by 5 days of reperfusion. These results, which are consistent with the findings of DiBona and Powell,23 indicate that development of necrosis is more likely related to tissue water content after storage than to the content after reperfusion. They also indicate that the increase in tissue water content during hypothermic storage is more likely related to development of myocardial necrosis than to graft survival after 5 days of reperfusion. Zikria and associates 24 reported that hydroxyethyl starch, an oncotic component in UW solution, minimizes differences in tissue water content and ionized potassium content between necrotic and normal areas, which results in low pathologic scoring. This supports the idea that tissue swelling correlates with the development of myocardial necrosis. Swanson and associates? reported that storage in UW solution suppressed the increase of the tissue water content of the myocardium after 12 hours of storage and subsequent reperfusion compared with Stanford and CM solutions, although no suppression was found after 5 hours of storage and subsequent reperfusion. Conversely, Yeh and associates'? reported that tissue water content after 6-hour storage in UW solution was lower than that in the Stanford solution, although tissue edema was induced after reperfusion. Our observations are similar to those of Yeh and associates.!? although the extent of edema in our present study was lower than that reported by their group. Discrepancies in these reports may be due to the fact that they used a crystalloid solution, whereas we used blood during reperfusion. Decrease in the yield of mitochondrial protein may be caused when (l ) the myocardium has been severely damaged by myocytolysis, resulting in loss of intracellular components, or (2) the mitochondria has structurally disintegrated (i.e., flocculation) and has behaved differ-

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 3

ently from intact mitochondriaduring isolationcentrifugation.P Becausea varied degree of mitochondrialdamage was possible, we estimated mitochondrial yields as yields of protein present in mitochondrialfractions. Similar yields of mitochondrial protein in control groups assuredus that no technicalvariation occurred in the isolation procedure. The yields of mitochondrial protein were not affected by ischemia but decreased after reperfusion for 1 day. This decrement was more significantin the CM groups than in the UW groups. However, no further decrease was induced in the following 4 days of reperfusion. This fact indicates that myocardial injury causinglossof mitochondrialprotein took placewithin24 hours after the start of reperfusion. The yields of mitochondrial protein correspondingly decreased with increased necrotic size. Intracellular levels of high-energy phosphate compoundsare regarded as an index to predict the condition of preserved hearts. Maurer, Swanson, and DeBoer9 reported that the intracellular level of ATP and total adenine nucleotides was significantly higher in hearts stored in UW solution than in those stored in CM solution. Ledingham and associatesl- reported that UW solution was overwhelmingly effective in preserving hearts with respect to mechanical performanceand ATP level at the end of the ischemicperiodand that high ATP level may be the effect of dehydration. Swanson and associates," using dog hearts, and Makowka and associates,II using rat hearts, indicated that hearts stored in UW solution under hypothermic conditions showedsignificantly higher intracellular ATP levels after reperfusioncomparedwith those stored in the Stanford solution. These facts imply that storage in the UW solution not onlyreduceslossof ATP but also protects the ATP synthesizing system from ischemic injury. It has been shown that mitochondrial oxidativephosphorylation is inhibited by ischemia.26,27 The present study showed that P /0 value and state 3 respiration rate remainedsignificantly higher in UW groups than in CM groups, indicating that the UW solution was capable of maintaining the oxidative phosphorylation activity; yet, an apparent but insignificant difference in state 4 respiration rate occurred between UW and CM groups. Mitochondrial respiratory functions are affected by various factors associated with ischemia and reperfusion processes, such as accumulation of fatty acids,28, 29 intracellular Ca 2+,30,31 and oxygen radicals.32.34 It has been reported that accumulated free fatty acids accelerate state 4 respiration, lower respiratory control index and P /0 values, and inhibit ATP synthesiswithout affecting state 3 respiration.P! 36 This is consistentwith the results of the present study with respect to inhibited phosphory-

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lation activity. Free fatty acids accumulating during ischemia might thus inhibit mitochondrial function; we did not, however, examine how albumin might have suppressed the action of free fatty acids on the function of isolated mitochondria. The precise mechanism that is responsible for inhibition of mitochondrial functions remains to be identified. The present study confirms the superiority of UW solutionoverCM solutionin preservingthe myocardium: UW solution is capable of suppressing tissue swelling during prolonged hypothermicischemia,which may contribute to reduced development of necrosis, protection of mitochondrial respiratory function, and maintenance of physiologic cellular integrity during reperfusion. We thank Drs. Masachika Kuwabara, Kenji Araki, and Hideharu Maruyama of Miyazaki Medical College for their helpful suggestions. We also thank Ms. Catherine C. Bryson, BA, forreading the manuscript andMs.Yuko Miyata forpreparing the manuscript. REFERENCES 1. Billingham ME, Baumgartner WA, Watson DC, et al. Distant heart procurement for human transplantation: ultrastructural studies. Circulation 1980;62(Suppl):Il 1-9. 2. Reitz BA, Brody WR, Hickey PR, Michaelis LL. Protection of the heart for 24 hr with intracellular (high K+) solution and hypothermia. Surg Forum 1974;25:149-51. 3. Belzer FO,Southard JH. Principles of solid-organ preservation by cold storage. Transplantation 1988;45:673-6. 4. D'Alessandro AM, Sollinger HW, Hoffmann RM, et al. Experience with Belzer UWcold storage solution insimultaneous pancreas-kidney transplantation. Transplant Proc 1990;22:532-4. 5. D'Alessandro AM, Kalayoglu M, Sollinger HW, et al. Experience with Belzer UWcold storage solution inhuman liver transplantation. Transplant Proc 1990;22:474-6. 6. Ploeg RJ. Kidney preservation with the UW and EuroCollins solutions. Transplantation 1990;49:281-4. 7. Swanson DK, Pasaoglu I, Berkoff HA, Southard JH, Hegge JO. Improved heartpreservation with UW preservation solution. J Heart Transplant 1988;7:456-67. 8. Wicomb WN,HillJD,Avery J, Collins GM.Optimal cardioplegia and 24-hour heart storage with simplified UW solution containing polyethylene glycol. Transplantation 1990;49:261-4. 9. Maurer EJ, Swanson DK, DeBoer L WV. Comparison of UW and Collins solution for preservation of the rat heart. Transplant Proc 1990;22:548-50. 10. YehT,HananSA,Johnson DE,etal.Superior myocardial preservation with modified UW solution after prolonged ischemia intherat heart. AnnThorac Surg1990;49:932-9. 11. Makowka L, Zerbe TR, Chapman F, et al. Prolonged rat cardiac preservation with UWlactobionate solution. Transplant Proc 1989;21:1350-2.

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