Superior qualities of University of Wisconsin solution for ex vivo preservation of the pig heart

Superior qualities of University of Wisconsin solution for ex vivo preservation of the pig heart The components of the University of Wisconsin solution have the potential to enhance and extend heart preservation. We have evaluated University of Wisconsin solution by comparing it with St. Thomas' Hospital cardioplegic solution in the isolated pig heart subjected to 8 hours of ischemia at 4° C (n = 6 in each). The hearts were perfused ex vivo with enriched autologous blood for the control and the postpreservation assessments. Morphologic, metabolic, and functional evaluations were performed. Left and right ventricular function as assessed by the slope values of systolic and diastolic pressurevolume relationships of isovolumically contracting isolated heart was better preserved by University of Wisconsin solution (percent reduction: left ventricular systolic, 52.4 % ± 5.5% versus 17.7% ± 6.7% [p < O.OOl~ right ventricular systolic, 125.6% ± 46.4% versus 65.5% ± 31.4% (p < 0.05]; right ventricular diastolic, 112.3% ± 48.7% versus 40.2% ± 31.3% fp < 0.02] after St. Thomas' Hospital and University of Wisconsin preservation, respectively). Postischemic recovery of left ventricular rate of rise of pressure and myocardial oxygen consumption were significantly improved after University of Wisconsin preservation (percent reduction, rate of rise of pressure: St. Thomas' Hospital 39.3 % ± 8.1 %; University of Wisconsin 18.1 % ± 4.6%; percent reduction, myocardial oxygen consumption St. Thomas' Hospital 55.1 % ± 6.9%, University of Wisconsin 24.8% ± 6.7%; p < 0.001). Microvascular functional integrity as assessed by coronary vascular resistance was weD maintained throughout the postischemic period and was similar to the preischemic control value in the University of Wisconsin group. By contrast, a significant increase was found at the beginning of postpresenation reperfusion, with a progressive rise thereafter in the St. Thomas' Hospital group (p < 0.001). Preservation of myocardial adenosine triphosphate was improved and energy charge was unchanged after 8 hours of ischemia and reperfusion in the University of Wisconsin-preserved hearts compared with the St. Thomas' Hospital-preserved hearts (p < 0.01). Electron microscopic examination revealed substantiaDy better preservation of the contractile apparatus after preservation with University of Wisconsin solution. Myocytes from hearts receiving University of Wisconsin solution, unlike those given St. Thomas' Hospital solution, showed relaxed myofibrils with prominent I-bands. We conclude that University of Wisconsin solution has the potential to improve the preservation of the heart" and possibly prolong the ischemic period in cUnical cardiac transplantation. (J THORAC CARDIOVASC 8URG

1992;104:229..40)

Pankaj S. Mankad, FRCSEd(C/Th),a,c Nicholas J. Severs, PhD,b David R. Lachno, DPhil,a,c Stephen Rothery, BSC,b and Magdi H. Yacoub, FRCS,a,c London and Middlesex, United Kingdom From the Departments of Cardiothoracic Surgery" and Cardiac Medicine," National Heart and Lung Institute, London, United

Kingdom, and Harefield Hospital, Middlesex, United Kingdom,"

Received for publication Aug. 7, 1990. Accepted for publication April 12, 1991.

Address for reprints: Magdi H. Yacoub, FRCS, Department of Cardiac Surgery, Harefield Hospital, Harefield, Middlesex UB9 6JH,

United Kingdom.

12/1/30414

Donor heart preservation by a solution that will ensure rapid and complete recovery of normal myocardial function is important for successful cardiac transplantation. Despite extensive research.l'! hypothermic storage after a single infusion of a cold cardioplegic solution is currently the only clinically used method for cardiac preservation. This technique offers up to 4 hours of relatively safe ischemic time, although it does not completely prevent the adverse effects of ischemia on myocardial function. A longer period of preservation may increase the donor

229

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Mankad et al.

Table I. Composition of solutions UWsolution

ST solution

KH2P0 4 (mmol/L)

25.0 NaCI (mmoljL) 5.0 KCI (rnmol/L) 5.0 MgCh (mmol/L) 3.0 CaClz (rnmol/L) 30.0 Procaine (mmolfL)

MgS04 (mmol/L)

Adenosine (rnmol/L) Glutathione (mmol/L) Raffinose (mmolfL) Allopurinol (mrnol/L) K lactobionate (mmoljL) Pentastarch (%) Insulin (IVIL) Osmolarity (mOsm/L) pH

144.0 20.0 16.0 2.4

1.0

1.0 100.0

5 40

320

7.4

300-320

5.5-7.5

organ pool, allow more time for tissue typing, and possibly shorten the transplant waiting lists. The recently introduced University of Wisconsin (UW) preservation solution has revolutionized clinical liver preservation.V and in experimental studies its superiority for the preservation of kidney and pancreas has been demonstrated.13, 14 However, the potential role of UW solution in cardiac transplantation has not been adequately defined. Therefore we decided to evaluate UW solutionby comparing it with 81.Thomas' Hospital (ST) cardioplegic solution, which is currently being used for myocardial preservation during routine cardiac operationsand transplantation. Theobjective ofthisstudywas to perform critical evaluationof UW solution compared with ST solution for the preservation of functional, metabolic, and morphologic aspects of the isolated pig heart.

Materials and methods Hearts were obtained from Yucatan pigs of both sexes and weighingbetween 20 and 35 kg each. After premedicationwith atropine sulfate (40 ~g/kg) and ketamine hydrochloride (10 mg/kg), the animals were anesthetizedwith intravenousadministration of propofol (3 mg/kg), intubated, and supported by a volume-controlled ventilator. Anesthesia was maintained with 60% oxygenand 400/0 nitrous oxide (4 to 6 L/min). Pancuroniurn bromide (0.08 mg/kg) was givenbefore the median sternotomy was done. While the chest was being opened, two units of blood (each unit approximately 500 ml) were collected via a cannula inserted into a femoral artery to prime the perfusion circuit. Intravenous fluids were administered through another cannula in a peripheral ear vein. Systemic blood pressure was maintained over a mean of 70 mm Hg throughout the procedure. Each pig was heparinized with 300 IV of beef lung heparin per kilogram of bodyweight,and the heart isolated,excised, and immediately immersed in cold (4 0 C) Ringer's solution. Prompt diastolic arrest wasachievedbyinfusing300 ml ofeither ST solution (David Bull Laboratories, Mulgrave, Victoria, Australia) or UW solution (E. I. DuPont de Nemours & Co., Inc., Bannockburn, Ill.) (n = 6 in each), cooledto 4 0 C, into the aortic root at a pressure of 50 mm Hg; this is the technique that we use in clinical cardiac transplantation.P The composition of the two solutions is shown in Table I.

While still immersed in cold Ringer's solution, intraventricular balloons (No. 11 Qualatex balloon, Pioneer Balloon Co., Wichita, Kan.), with a complianceof 65 ml, were connectedto a pressure transducer and sutured to the tricuspid and mitral valveanuli. The left ventricle was vented by a small apical stab incision. A thermistor probe was inserted into the interventricular septum for continuous monitoring of myocardial temperature. The aorta was clamped, a cannula was inserted into the innominate artery, and both the aorta and the cannula were deairecl. The heart was then mounted. on a retrograde perfusion apparatus, consisting of a Watson-Marlow roller pump (Smith & Nephew Watson-Marlow, Cornwall, England) and a Capiox 300hollow-fiber oxygenator (Terumo Corp., Somerset,N.J.) with integrated heat exchanger, via a cannula in the innominate artery, and placed inside an insulated glass cylinder. It was immediately perfused in a nonpulsatileretrograde fashionwith enriched autologous blood (hematocrit 28% to 300/0), containing n-ribose (1 grri/L), insulin (20 lUlL), and glucose (14.25 mg/rnin), at an aortic pressure of 80 mm Hg and perfusate temperature of 37° C. The mean period between the arrest and the reperfusion was 8.3 ± 2.6 minutes. After 20 minutes of perfusion,control data regarding functional and metabolicstate of the hearts were obtained. The hearts were then rearrested with 600 ml of the same solutionused previously (that is,either ST or UW solution) and stored for 8 hours at 4 0 C. The ST solution-arrested hearts were stored, immersed in Ringer's solution, and the hearts preserved by UW solution were kept immersed in the UW solution.At the end of the periodof ischemia, intraventricular balloons were reinserted and the hearts perfused with the second unit of enriched autologousbloodfor 2 hours. Repeat data for the functional and the metabolicstates were collected at half-hour intervals. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared bythe National Academy of Sciencesand published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Functional and metabolic assessment. So that the degreeof tissue edema could be assessed, the hearts were weighedimmediately after the first arrest, and this weight was compared with the weight at the end of ischemia and at the end ofreperfusion. Coronary flow was measured continuously by on-line electromagnetic blood flow probe (Nihon-Kohden Corporation, Tokyo, Japan). From these measurements, coronary vascular resistance(CVR) was calculated by the following formula:perfusion pressure (mm Hg) multiplied by heart weight (gm) divided by coronary flow (rnl/rnin). Functional evaluation of the isovolumically contracting hearts was performed aided by the previouslyintroduced intraventricular balloons. The zero volume for isovolumic ventricular contraction, defined as the volume at which the ventricle generates a diastolicpressure of 1 to 2 mm Hg, wasdetermined for both the ventricles by introducing a measured quantity of normal saline into the balloons. They were then sequentially inflated with 10, 20, 30, and 40 ml of saline. At each balloon volume, transballoon left ventricular systolicand diastolicpressure and right ventricular systolic and diastolic pressure plus rate of left ventricular pressure rise (dP/dt) were recorded. At the balloonvolumeof 10and 40 ml, samples of arterial and coronary sinus blood were collectedfor the measurement of blood gases.From these values,oxygencontent wasdetermined by the

Volume 104

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August 1992

250

231

1s 1

200. CJl

I

E

T

150

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Fig. 1. A and B, Left ventricular systolic (S) and diastolic (D) pressure-volume (P- V) relationship of isovolumically

contracting isolated pig heart before (open circles) and after (open triangles) 8 hours of preservation with ST cardioplegic solution (A) or UW solution(B)(n = 6 in each). *p < 0.01; **p < 0.001. ap < 0.05;"» < 0.001 compared with A.

method described by Behar and Severinghaus.!" Myocardial

oxygen consumption (MV0 2 ) was calculated from the following equation: MV02 = CBF (A - V), where CBF = coronary blood flow, A = arterial oxygen content, and V = the oxygen

contentof the coronary sinus effluent.Values were expressedas microlitersper 100gm per beat to normalize for heart weight and heart rate. Nucleotide content and electron microscopy. For determination of control myocardial nucleotidecontent and for baseline electronmicroscopy, full-widthbiopsyspecimenswere obtained with use of a Tru-Cut needle (Baxter Healthcare Corp., Pharmaseal Division, Valencia, Calif.) fr.om the in situ beating hearts. Both these investigations were repeated on similar biopsy material taken from the hearts after an 8-hour preservation period (that is, before reperfusion). Biopsyspecimenswere also obtainedat the end of the experiment (that is, after reperfusion) for the measurement of nucleotidecontent. Data were obtained at the end of ischemia and at the end of reperfusion and compared with each other and with preischemic control values.

Energycharge, a quantita tiveestimate of the energy state of the cell,'? was calculated by the formula ATP + V2 ADP/ (ATP + ADP + AMP), where ATP, ADP, and AMP are

adenosine triphosphate, diphosphate, and monophosphate. For morphologic assessment, the biopsy specimens were immersed in 2.50/0 glutaraldehyde in sodium cacodylate 0.1 mol/L (pH 7.4) and cut into 1 rnm? blocks. After further fixation in the glutaraldehyde solution for 2 hours, the samples were rinsed in cacodylate buffer and postfixed in 2% osmium tetroxide (cacodylate buffered) for 1 hour. The samples were then washed in 30% ethanol, en bloc stained in saturated uranyl acetate in 500/0 ethanol, dehydrated in ethanol, and embedded in Emix epoxy resin (Fisons Polaron, Loughborough, United Kingdom). Polymerization was done at 60° C. For screeningof the samplesby light microscopy, semithin (0.5 to 1 ~m) sections werecut with a Reichert Ultracut E ultramicrotome (Leica Inc. ~ Deerfield, Ill.) and stained with 0.1 % toluidine blue in 10/0 borax/50% ethanol. For electron microscopy, thin sections, prepared with the ultramicrotome, were stained in saturated

232

The Journar of Thoracic and Cardiovascular Surgery

Mankad et at.

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30

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Fig. 2. A and B, Right ventricular systolic (S) and diastolic (D) P- V relationship of isovolumically contracting isolated pig heart before (opencircles) and after (opentriangles) 8 hours of preservation with ST cardioplegic solution (A) or UW solution (B) (n = 6 in each). *p < 0.01; **p < 0.001; o.p < 0.02 compared with A. 70 ~

UO:

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Fig. 3. Coronary vascular resistance (CVR) at control (C), initial postischemic reperfusion (R/), and end of reperfusion (R2) in isolated pig heart subjected to 8 hours of preservation with 81. Thomas' Hospital (ST) cardioplegic solution (filled bars) or University of Wisconsin (UW) solution (hatchedbars) (n = 6 in each). *p = not significant; **p < 0.02; fXp < 0.05; aap < 0.01 compared with UW.

Volume 104

Number 2

UW solution for heart preservation

August 1992

50 40 c

_

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~

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233

30

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HYPOXANTHINE

Fig. 4. Myocardial content of ATP (adenosinetriphosphate), ADP (adenosinediphosphate),and AMP (adenosine monophosphate) (A)and of adenosine,inosineplus hypoxanthine(B)at control (C), end of ischemia (EOL), and end of reperfusion (EOR) in isolated pig heart subjected to 8 hours of preservation with ST or UW solution (n = 6 in each), *p < 0.01; **p < 0.001. uranyl acetate (in 50% ethanol), followed by Reynold's lead citrate, and examined with a Philips EM 301 electron microscope(Philips Electronic Instruments Inc., Mahwah, N.J.). For measurement of nucleotidecontent, the specimenswere snap frozen and stored in liquid nitrogen until submitted to the process of extractionwith perchloricacid, 0.6 mol/L, The analysis for nucleotidecontent was performed by the reversed-phase high-performance liquidchromatography method as described in detail previously.l! Expression of results. From the values of transballoon systolic and diastolic pressures obtained at different balloon volumes, pressure-volume (P-V) curves were plotted for both the right and the left ventricles. The slopevaluesof these curves were analyzed for statistical significance. The slope values of diastolic P-V curves were obtained by plotting a regression line through the P-V points. Differences in the preischemic and postischemic valuesof dP Jdt and myocardialoxygenconsumption were expressed as a percentage reduction over the control values, whereasthe absolutevalues of CVR were plottedfor the preischemic control hearts and for the beginningand the end of

postischemic reperfusion. We used Student's paired t test to assess the levelof statistical significance between the preischemic control and the postischemic recoverydata within a group (that is, ST or UW), and we used the unpaired t test to compare the differences betweenthe groups. For the latter comparison,the postischemic valueswere expressedas a percentage of their controlvalues.Significance wasassumed when the p value was less than 0.05. All values are expressed as the mean ± standard deviation.

Results Functional studies. There was no statistically significant difference in the postpreservation data obtained after lh, 1, 1V2, and 2 hours of reperfusion. The mean values were used for analysis. Fig. 1 shows the left ventricular systolic and diastolic P-V relationship in the preischemic control heart and after postischemic reperfusion in the ST group (Fig. 1, A) and the UW group (Fig. 1, B). In the

The Journal' ofThoracic and Cardiovascular

234 Mankad et al.

Surgery

Table II. Energy charge ofpreserved hearts Solution

Control

EO!

EOR

ST (n = 6) UW (n = 6)

0.850 ± 0.02 0.838 ± 0.05

0.748 ± 0.014* 0.80 ± 0.02

0.785 ± O.035t 0.838 ± 0.04

ST, 81. Thomas' Hospital cardioplegic solution; UW. University of Wisconsin solution; EOI, end of ischemia; EOR, end of reperfusion. *p < 0.001. tp < 0.01.

ST group the slope value of the systolic P- V curve diminished from 4.54 ± 0.57 to 2.1 ± 0.18 (p < 0.001), and in the UW group the reduction, although less (from 4.55 ± 0.49 to 3.74 ± 0.49), was still significant (p < 0.01). The percentage reduction in the UW group was less than in the ST group (UW 17.7% ± 6.70/0; ST 52.4% ± 5.50/0; p < 0.001). Similarly, chamber stiffness, defined as a change in pressure relative to a change in volume, increased after preservation. In the ST group the increase was from 1.13 ± 0.28 to 2.6 ± 0.62 mm Hg/rnl (p < 0.01). In the UW group the change was from 1.33 ± 0.39 to 2.19 ± 0.49 mm Hgjml (P < 0.01). The percentage increase in the UW group (65.5% ± 31.40/0) was significantly less (p < 0.05) than that in the ST group (125.6% ± 46.4%). Slope values of right ventricular systolic curves in both the groups were similar. However, right ventricular chamber stiffness increased by 40.2% ± 31.30/0 after UW preservation compared with an increase of 112.30/0 ± 48.70/0 in the ST group (p < 0.02) (Fig. 2). There was a reduction in dP /dt and in myocardial oxygen consumption after 8 hours of preservation in both the groups. The hearts perfused with UW solution showed significantly better recovery of dP jdt and myocardial oxygen consumption than the hearts perfused with ST solution (p < 0.001). The UW-perfused hearts showed reduction in dPjdt from 1906.7 ± 81.1 mm Hg/sec to 1560.8 ± 87.4 mm Hgjsec (p < 0.001), whereas in the ST hearts the reduction was from 2070 ± 211.7 to 1250 ± 160.5 mm Hgjsec (p < 0.001). The percentage reduction in UW-perfused hearts (18.10/0 ± 4.6%) was significantly less than in the ST group (39.3% ± 8.1 %; p < 0.001). In the control assessment, as the work performed by the hearts was increased by changing the balloon volume (from 10 to 40 ml), myocardial oxygen consumption also increased in parallel (ST from 40.8 ± 2.3 to 74.4 ± 11.6Jll/lOOgm/beat;UWfrom44.5 ± 5.7 to 86.7 ± 9.1 ~1/100 gmjbeat). However, there was little change in the postischemic oxygenconsumption in the ST hearts by varying the balloon volume (29.7 ± 3.2 at 10 ml and 32.4 ± 4.8 at 40 mI). This was less than in the control hearts (p < 0.05 at 10 ml andp < 0.001 at 40 ml). In the UW group, although there was slight overall

reduction in myocardial oxygen consumption, the increase associated with increasing balloon volume was still well maintained (at 10 m139.1 ± 4.2, P > 0.05; at 40 ml 64.8 ± 7.9, p < 0.05). The mean percentage reduction in myocardial oxygen consumption in UW-perfused -hearts was 24.8% ± 6.7%,whereas in the ST group it was 55.1% ± 6.9% (p < 0.001). Weights of the hearts in the ST group, after the initial arrest, were significantly higher than in those of the UW group (ST 201.5 ± 51.1, UW 165.8 ± 37.5; p < 0.01). Even though there was no difference in the preischemic and postischemic weights of the hearts in either group (ST: preischemic 201.5 ± 51.1, postischemic 196.4 ± 43.9; UW: preischemic 165.8 ± 37.5, postischemic 173.8 ± 34.2), there was an increase in the postischemic CVR in the ST-perfused hearts (preischemic 40 ± 3.8, postischemic 46.4 ± 8.5), but this failed to reach statistical significance. In these hearts CVR continued to rise and was 54.6 ± 9.5 by the end of a 2-hour perfusion period (p < 0.02). In the UW group CVR was well maintained throughout the period of postischemic assessment and was similar to the preischemic control value (Fig. 3). Metabolic studies. The mean contents of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate for both groups at control, end of ischemia, and end of reperfusion are shown in Fig. 4, A. There was no difference in the preischemic control values of adenine nucleotides (in nmol/Lzmg protein) between the two groups (adenosine triphosphate: ST 34.5 ± 2.81, UW 35.5 ± 5.66; adenosine diphosphate: ST 11.6 ± 2.23, UW 11.3 ± 1.79; adenosine monophosphate: ST 1.5 ± 0.5, UW 1.3 ± 0.14). At the end of the ischemic period the values were as follows: adenosine triphosphate: ST 24.1 ± 3.91, UW 27.3 ± 3.29; adenosine diphosphate: ST 13.3 ± 1.78, UW 9.4 ± 2.14; adenosine monophosphate: ST 3.6 ± 0.69, UW 3.2 ± 0.97. The differencebetween the groups was not significant. However, at the end ofreperfusion the adenosinetriphosphate levels were significantly better in the UW-preserved hearts than in the ST-preserved hearts (ST 14.8 ± 3.92; UW 23.2 ± 1.82; p < 0.01). The mean contents of adenosine, inosine, and hypox-

Volume 104 Number 2 August 1992

UW solution for heart preservation

235

Fig. 5. Cardiac muscle cell ultrastructure from a control heart. A, Surveyview. B, Higher magnification shows good preservation ofcellular structure. Notethatmyofibrils (My) areina stateofcontraction, andmitochondria (M i) have intact cristae and dense matrices. Z, Z-band; N, nucleus; nu, nucleolus; T, transverse tubule. (Original magnifications: A, X5700; B, X30,500.) anthine for each group at preischemic control, end of ischemia, and end of reperfusion are compared in Fig. 4, B. There was a marked increase in the contents of the three nucleotides over the control values at the end of ischemia in the UW group compared with the ST group. The control values for adenosine, inosine, and hypoxanthine, respectively, in the two groups were as follows: ST 0.05 ± 0.12, 0.7 ± 0.32 , and 0.4 ± 0.23; UW 0.05 ± 0.08,0.6 ± 0.12, and 0.5 ± 0.17. On cessationof ischemia, the control values for adenosine, inosine, and hypoxanthine were as follows: ST 0.08 ± 0.08, 1.8 ± 1.1, and 0.58 ± 0.27;UW 1.3 ± 0.57 (p <0.01), 8.0 ± 1.17 (p < 0.001), and 1.5 ± 0.43 (p < 0.01). The calculated adenine nucleotide energy charge is shown in Table II. Energy charge remained well maintainedin the UW group (controlvalue 0.838 ± 0.05; end of ischemia 0.80 ± 0.02; end of reperfusion, 0.838 ± 0.04), but in the ST group there was a significant reduction (control value 0.85 ± 0.02; end of ischemia

0.748 ± 0.014 (p < 0.001) ; 0.785 ± 0.035 (p < 0.01)).

end

of

reperfusion,

Myocardial ultrastructure. Ultrastructural examination revealedconsiderablevariation in the preservationof cellularcomponents withineach biopsyspecimen.For the similaritiesand differences betweenthe controlspecimens and different postpreservation conditions to be assessed reliably, it was therefore necessaryto examine (1) biopsy specimens from six hearts for each of the experimental conditions, (2) several blocks of embedded tissue from each biopsy specimen, and (3) multiple sections cut at different levels within each block. The ultrastructure of the myocardium of control samplesis illustrated in survey view in Fig. 5, A , and at higher magnification in Fig. 5, B. The contractile apparatus generally appeared well organized and aligned, with a clearly defined sarcomeric banding pattern (Fig. 5, A). The I bands were not prominent, indicating that the myofibrils werein a state of contraction, although this was

236

Mankad et al.

The Journal of Thoracic and Cardiovascular Surgery

Fig. 6. Cardiac muscle cell ultrastructure from a heart a fter 8 hours of preservation with ST cardioplegic solution. A, Survey view. B, H igher magnification. Z , Z-band. Note that nuclei (N ) show clumping (c) and margination (m) of chromatin, clearing of background nucleoplasm, and widening of perinuclear space (p). In addition, mitochondria (Mi) show clearer matrices and some loss of cristae (*) compared with views in Fig. 5. (Original magnifications: A, X5700; B, X25,500.)

generally within the normal physiologicrange. Mitochondria were well preserved, showing a normal uniformly dense matrix and well-defined inner and outer membranes with compactly organized cristae. Membranes of the transverse tubules and sarcoplasmic reticulum were readily identified in the usual configurations . Nuclei were also characteristically well preserved (Fig. 5, B). The nuclear contents appeared predominantly as diffuse chromatin, often with a conspicuous nucleolus, and were bounded by the evenly spaced double membranes of the nuclear envelope. After 8 hours of preservation with ST cardioplegic solution, overall preservation of myocyte structure remained similar in many respects to that seen in the control hearts, although some signs of deterioration were consistently observed (Fig. 6). The most obvious adverse changes were in the mitochondria and nuclei. Mitochondria typically had a paler matrix than in control hearts,

and some disruption of cristae was common (Fig. 6, B). The occasional severely affected mitochondria appeared empty and swollen. The nuclei characteristically revealed a greater proportion of condensed chromatin than was observed in the control hearts , and a reduction in density of the background nucleoplasmic matrix was apparent. Margination of the heterochromatin was common, and the perinuclear space between the membranes of the nuclear envelope was frequently dilated . The contractile apparatus for the most part was in a state of contraction with short or absent l-bands. Cytoplasmic matrix, transverse tubules, sarcoplasmic reticulum, and other components of the cell appeared similar to those observed in the control hearts . Biopsy specimens from the UW-preserved hearts showed similar changes in myocyte mitochondria and nuclei to those in the ST-preserved hearts, although these changes were less severe. Some disruption of mitochon-

Volume 104 Number 2 August 1992

UW solution for heart preservation

237

Fig. 7. A and B, Cardiac muscle cell ultrastructure from a heart after 8 hours of preservation with UW solution . Note excellent preservation ofmyofibrils (My). Prominent l-bands (I) demonstrate that, in contrast to the cells in Fig. 6, the contractile apparatus is in a state ofrelaxation. Mitochondria (Mi) and nuclei (N) show similar structural features to those of hearts receiving ST cardioplegic solution (Fig. 6), although the nuclei are more elongated in overall shape because of the relaxed state of the cells. Z, Z-band; nu, nucleolus; sr, sarcoplasmic reticulum; ID, intercalated disc. (Original magnifications : A, X5700; B, X30,500.)

drial cristae and loss of matrix density, together with chromatin clumping and margination in the nuclei, was nevertheless observed (Fig. 7). However, a striking difference was apparent in the configuration of the contractile apparatus. Myocytes in the UW-preserved group consistently showed relaxed myofibrils with prominent I-bands (see Fig. 7). All features ofthe organization of the contractile apparatus consistently appeared well preserved. Other components of the cell appeared as in the control and the ST experiments. Discussion This study has demonstrated that a single infusion of UW preservation solution provides significantly better morphologic, metabolic, and functional preservation of the isolated pig heart subjected to 8 hours of hypothermic ischemic arrest at 4 0 C than can be achieved with the ST

solution. Our findings are in agreement with the recently reported superiority of UW solution in preserving isolated canine and rat hearts. 19, 20 The present study also shows that UW solution fails to achieve complete recovery of myocardial function after 8 hours of global ischemia in the pig heart, thus highlighting the need for further research in this area. Functional assessment. Ex vivo assessment of ventricular function in various animal models has been done by several investigators using different techniques." No particular method has been considered entirely satisfactory. It has been shown that the P-V relationships of ejecting and isovolumically contracting ventricles are virtually identical 22, 23 and that the slope value of isovolumic systolic P-V curve, which is linear under physiologic conditions, is directly related to the contractile state of the myocardium. 24, 25 It is also considered to be a reliable

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method for assessment of diastolic function.i" Therefore we have used this technique to assess ventricular function. In the experiments described, since each heart acted as its own control, the effect of geometric conditions, that is, anatomic size, shape, and muscle mass, on the P- V relationship was nullified. The presentation of a complete P-V diagram (systolic and diastolic P-V curve), as shown here, permits a synopsis of the important functional data and is one of the most unambiguous indices of ventricular function. Furthermore, the area under the systolic and the diastolic P- V curve has a dimension of work (energy) and can be correlated with myocardial oxygen consumption.i? Many previous investigators have used the function of the heart in vivo as a control assessment. This disregards the effect of ischemia between harvesting and storage and also does not take into account the entirely different experimental conditions that exist in in vivo control assessment and in ex vivo postpreservation assessment. To obviate this we used an ex vivo perfusion system to assess left and right ventricular function after harvesting and immediately after the preservation period. Moreover, this preparation allows for the assessment of right ventricular function, which is equally important, especially in the immediate postoperative period after cardiac transplantation when the recipient often has high pulmonary vascular resistance." Under these conditions we have shown that left ventricular systolic function and both right and left ventricular diastolic functions were better preserved by UW solution than with ST solution. UW solution. The composition of the two solutions differs considerably. UW solution is an intracellular type of solution with a high concentration of potassium (125 mrnol/L) and a relatively low concentration of sodium (20 mmoljL). A high concentration of potassium and a low concentration of sodium have been shown to cause a prominent increase in CVR in rat hearts at 4 0 C 29 and to elevate the resting tension in the guinea pig myocardium. 30 In our study UW solution was not overtly detrimental the myocardium or to coronary vasculature. This could be due to the complex ionic interactions among sodium, calcium, and potassium at deep hypothermia since high CVR after high potassium and low sodium has been attributed to the activation of the slow calcium channel and the sodium-calcium exchange mechanism. In the rat experiments cited,29 infusion of hearts with intracellular solution after washout of calcium from the coronary vasculature did not increase CVR. It is conceivable that the absence of calcium from UW solution might have a protective effect on the coronary vasculature. Furthermore, other adverse effects of potassium in excess of 30 mmol/L, namely, intracellular calcium sequestration and accelerated degradation of adenine nucleotides in the rat heart, as reported by Hearse, Stewart, and Braim-

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The Journal of Thoracic and Cardiovascular Surgery

bridge" have been shown at normothermia. It is possible that deep hypothermia affords protection against the adverse effects of hyperkalemia. UW solution contains impermeants in the form of raffinose, a nonmetabolizable trisaccharide, and lactobionate, an anion that substitutes for the freely permeable chloride ion. These components provide an extracellular osmotic force that has been shown to limit hypothermic cell swelling in the kidney, pancreas, and liver. 32 In addition, pentastarch, a type of hydroxyethyl starch, provides a colloidal oncotic pressure that will limit the expansion of the interstitial space. In contrast, ST solution is purely a crystalloid, and, although ofa similar osmolarity to UW solution, it does not contain an oncotic agent. Hyperos- motic perfusates have been shown to improve ultrastructural and functional preservation of the isolated canine heart. 33,34 In this study weights in the ST hearts were significantly higher than in the UW hearts after the initial arrest, suggesting that the ST group was made edematous by the initial arrest. There was no difference in prepreservation and postpreservation weight of the hearts in either group, possibly suggesting no net gain of water and sodium. However, the possibility of intracellular edema formation resulting from hypothermia-induced inactivation of the sodium/potassium/adenosinetriphosphatase system35 cannot be excluded in the group of hearts preserved by ST solution, since on postpreservation reperfusion these hearts had significantly higher CVR and more deterioration in diastolic function than the UW group. The role of the pentafraction in preservation is still uncertain because in two recent studies equally good preservation of rat livers and kidneys was obtained by omission of the hydroxyethyl starch from UW solution. 36, 37 In the present study adenosine triphosphate appeared to be better preserved by UW solution than by ST solution, which does not contain adenosine. The extremely high concentration of inosine and elevated levels of adenosine and hypoxanthine at the end of ischemia in the UW-preserved hearts suggest that adenosine from UW solution is taken up and metabolized by myocytesduring the period of preservation. Inosine and hypoxanthine are known to stimulate the formation of adenine nucleotides via the "salvage pathway" through the generation of inosine monophosphate.P: 39 This could account for the better preservation of adenosine triphosphate in the UW group. Restitution of the myocardial adenosine triphosphate pool after ischemia has been shown to be associated with improved heart function.t? Adenosine and phosphate in the UW solution have been shown to stimulate adenosine triphosphate synthesis in the hypothermically perfused canine kidney."! However, adenosine has not been considered as an essential component of UW solu-

Votume 104

Number2 August 1992

tionfor the preservationof rabbit liver. 42 Oxygen-derived free radicals are thought to play an important role in causing endothelial and myocardial injury during ischemia and reperfusion.f- 44 Addition to antioxidants (superoxide dismutase plus catalase, allopurinol, and reduced glutathione) to cardioplegic solutions has been shownto be associated with improved recoveryof postischemicfunctionof the rat hearts.45-47 In the light of these studies, it is conceivable that the presence of both allopurinol and glutathione in UW solutions may also have contributed to its superior qualities. This is supported by our finding of maintenance of near normal CVR in the UW-preserved hearts as opposedto the ST hearts, which showedprogressive increase in CVR during postischemic reperfusion. This rise could be due to endothelialdamage caused by free radical-mediated reperfusion injury." Morphologic assessment. Ultrastructural changes could bedue to anoxia or to reperfusion injury.49-51 Electron microscopy reveals definite changes after 15minutes of anoxia. The earliest changes are seen in mitochondria, wherelocal swelling and decrease in matrix density occur at 15 minutes, and obviouschanges are seen in the nucleus and cytoplasm by 30 minutes. Further abnormalities in myocyte and capillary ultrasructure follow rapidly. Therefore the observed superiroity of UW solution in preserving myocardial ultrastructure suggests a time-related phenomenon. Although the morphologicchanges in mitochondria are similar in the two groups, near normal maintenanceof energycharge by UW solutionhighlights the better preservation of mitochondrial function during ischemia and after reperfusion. In conclusion, this study has shown that UW solution, when compared with currently used myocardial preservationsolution, has the potential to improvethe functional recovery and to prolong the ischemicperiod in clinical cardiactransplantation. These data havestimulated us to undertake a controlled trial of the UW solution,comparing it with the ST solution in our clinical cardiac transplantation program. We thank Dr. R. Mantell for his excellent help with functional evaluation, Mr. D. Dopson for the help with the animals, Mr. T. A. E. Sopwith and Mr. B. J. M. Bridgewater for perfusate analysis, and Mr. M. Collins for technical help.

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