Long-term preservation of the mammalian myocardium

J

THORAC CARDIOVASC SURG

1991;102:235-45

Long-term preservation of the mammalian myocardium Effect of storage medium and temperature on the vulnerability to tissue injury Human heart preservation for transplantation commonly involves infusion of cold cardioplegic solutions

and subsequent immersion in the same solution. The objectives of the present study were (1) to establish the temporal relationship between storage time (at 10° C) and the postischemic recovery of function in the isolated rat heart, (2) to assess, by metabolic and functional measurements, whether storing the heart in fluid as opposed to moist air had any effect on the viability of the preparation, and (3) to ascertain the optimal storage temperature. Isolated rat hearts (at least 6 in each group) were infused for 3 minutes with St. Thomas' Hospital cardioplegic solution No.2 at 10° C, stored at 10° C for 6, 12, 18, or 24 hours, and then reperfused at 37° C. Mechanical function, assessed by construction of pressure-volume curves (balloon volumes: 20, 40, 60, 80, 100, and 120 ILl), was measured before ischemia and storage and after 60 minutes of reperfusion. Function deteriorated in a time-dependent manner; thus at a balloon volume of 60 ILl the recovery of left ventricular developed pressure was 84.2 % ± 5.3 % after 6 hours (p = not significant when compared with preischemic control); 69.1 % ± 3.3% after 12 hours (p < 0.05); 55.6% ± 4.4% after 18 hours (p < 0.05), and 53.0% ± 6.8% (p < 0.05) after 24 hours of storage. Other indices of cardiac function, together with creatine kinase leakage and high-energy phospbate content, supported these observations. Since the recovery of the left ventricular developed pressure balloon volume curves were essentially flat after 18 and 24 hours of storage, either 6 or 12 hours of storage were therefore used in subsequent studies. Comparison of storage environment (hearts either immersed in St. Thomas Hospital cardioplegic solution No. 2 or suspended in moist air at 10° C for 6 or 12 hours) revealed no significant differences in functional recovery between the groups. Thus hearts recovered 94.9% ± 3.5% and 113.7% ± 12.4%, respectively, after 6 hours of storage and 71.6% ± 2.4% and 54.2% ± 7.9%, respectively, after 12 hours of storage. Enzyme leakage and tissue water gain were also similar in both groups of hearts. Finally, hearts (n = 6 per group) were subjected to 12 hours' storage at 1.0°, 5.0°, 7.5°, 10.0°, 12.5°, 15.0°, and 20.0° C. At a balloon volume of 60 J.LI, the postischemic recovery of left ventricular developed pressure was 50.0% ± 9.3%, 60.5% ± 5.2%, 53.9% ± 3.3%, 55.7% ± 2.2%, 24.0% ± 4.4%, 21.6% ± 6.3%, and 4.8% ± 3.4%, respectively. These results suggest that the optimal temperature for extended storage is between 1.0° and 10.0° C and tbat edema formation was not exacerbated by storage in cardioplegic solution. We have selected 7.5° C as the optimal hypothennic temperature for use in further studies to assess myocardial preservation during extended storage.

Akihiko Takahashi, MD, Mark V. Braimbridge, FRCS, David J. Hearse, PhD, and David J. Chambers, PhD, London. England

From Cardiovascular Research (Surgical Cytochemistry), The Rayne Institute, St. Thomas' Hospital, London, England. Supported in part by grants from the National Heart and Lung Institute (HL3945-01) and St. Thomas' Hospital Research Endowments Fund. Akihiko Takahashi was a STRUTH International Research Fellow.

Received for publication Oct. 17, 1989. Accepted for publication April 17, 1990. Address for reprints: David J. Chambers, PhD, Cardiovascular Research (Surgical Cytochemistry), The Rayne Institute, St. Thomas' Hospital, London SEI 7EH, UK.

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Takahashi et al.

The introduction of cold chemical cardioplegia 1 has increased the safety of cardiac operations. Most cardioplegic solutions, however, were developed for use in routine cardiac operations, in which ischemic times in excess of 2 to 3 hours were rarely required. With the increased time constraints of cardiac transplantation, cardioplegic solutions have been adopted for use as protective agents during harvesting, transport, and transplantation. A maximum time limit of 4 hours is usually adopted for storage- 3 because reliable recovery of the heart cannot otherwise be achieved. The logistics of cardiac transplantation would be enhanced if safe preservation times could be extended beyond this 4-hour time limitation. In the literature there are several reports"!' of experimental studies into extended preservation times for hearts from the dog, rat, rabbit, and other species. As long ago as 1965 Kondo and coworkers" described heart storage in hyperbaric oxygen for 12 and 24 hours with successful orthotopic transplantation. In 1968 Proctor and Parkers reported successful recovery (in vivo) of the dog heart after 72 hours of in vitro storage. In 1969 Freemster and Lillehei" reported successful implantation of canine hearts that had previously been preserved for up to 24 hours. Extended preservation of the mammalian heart is, therefore, possible, but limited clinical success to date may relate to the fact that routine cardioplegic solutions, designed specifically for short-term preservation, were usually employed. Since these solutions pay relatively little attention to some of the problems associated with long-term tissue storage (e.g., progressive and severe water gain that may be exacerbated by long periods of immersion in a crystalloid solution), it is perhaps not surprising that they have achieved limited success. In the present study, intended as a prelude to studies aimed at refining a solution specifically for long-term preservation, the isolated perfused rat heart preparation and the St. Thomas' Hospital cardioplegic solution No.2 (STH2) were used to define the relationship between myocardial preservation and the duration and temperature of storage and the effect of storing hearts in moist air as opposed to fluid during ischemia. Materials and methods Hearts. Hearts were obtained from male rats (240 to 300 gm body weight) of the Wistar strain (Bantin and Kingman). All animals received humane care in accordance 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 by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No 85-23, revised in 1985).

Table I. Composition ofperfusion medium and cardioplegic solution Krebs-Henseleit bicarbonate Compound NaCI NaHC0 3 KCI KH2P04 MgS04 MgCIz CaCI2

Glucose pH Osmolarity (mOsm H20)

buffer'? (mmolfl.} 118.6 25.0 4.70 I. J8 1.20 1.4

St. Thomas' Hospital cardioplegic solution! (mmolil.; 110.0 10.0 16.0

16.0 1.2

Il.l 7.4 316

7.8 324

Experimental preparation. The isolated perfused rat heart preparation, based on that described by Langendorff!" and described in detail elsewhere, 1 was used for this study. In this preparation oxygenated perfusion fluid (at 37° C) was infused into the aorta from a reservoir located 100 cm above the heart. The standard perfusate used throughout was Krebs-Henseleit bicarbonate buffer," pH 7.4 (when gassed at 37° C with 95% oxygen and 5% carbon dioxide), the composition of which is shown in Table I. A small fluid-filled balloon, attached to a cannula, was inserted through the mitral valve into the left ventricle. The cannula was attached to a pressure transducer via a three-way tap so that the balloon could be accurately filled with any required volume of fluid. Incremental balloon loading was achieved with the use of a 250 Jil syringe. In all studies the region of the sinus node was excised and right ventricular pacing (300 beats/min) was initiated during preischemic and postischemic aerobic perfusion. Global ischemic arrest was induced by clamping the aortic cannula. Short periods of preischemic infusion with cardioplegic solution (at 37° C or any desired degree of hypothermia) were achieved via a reservoir located 60 em above the heart and attached to a sidearm of the aortic cannula. For this study the STH2 was used, the composition of which is shown in Table I. All solutions used in this study were filtered through a 5.0 Jim porosity filter before

use."

Experimental time course. After ether anesthesia and intravenous injection of 200 IU heparin, the heart was rapidly excised, attached to the perfusion apparatus via an aortic cannula, and aerobically perfused for 30 minutes, during which time control values of various indices of cardiac function were measured (see next section, "Indices Measured and Expression of Results"). Hearts were then arrested by a 3-minute infusion of cardioplegic solution (at the degree of hypothermia desired) and maintained in a globally ischemic state for various durations before 60 minutes of aerobic reperfusion. Throughout the reperfusion period coronary effluent was collected for measurement of creatine kinase leakage. I? After 60 minutes of reperfusion all indices of cardiac function were again measured, and the hearts were then freeze-clamped 18 at -197° C and stored before analysis of myocardial high-energy phosphate content. Three variables, as discussed in the paragraphs that follow, were studied.

Volume 102 Number 2 August 1991

237

Long-term myocardial preservation

Table II. Effect of 6-, 12-, 18-, and 24-hour global hypothermic (10.0 C) storage on postischemic functional 0

recovery. creatine kinase leakage, myocardial water content, and high-energy phosphate (ATP and CP) content of hearts* Control (preischemic value) LVDP (em H20) LVEDP (em H20) CK leakage (lU/60 min/gm dry wt) Total reperfusate volume (mil Wet/dry wt ratio ATP content (urnol/grn dry wt) CP content (umol/gm dry wt)

Postischemic recovery aftervarying durations of ischemia (hr)

(n = 24)

6 (n = 6)

12(n=6)

125.9 ± 5.7 3.6 ± 0.6

106.0 ± 6.7 6.5 ± 2.1 119.5 ± 25.3

87.5 ± 3.7t 52.5 ± 9.7 292.5 ± 45.1

70.0 ± 5.4t 91.7 ± 8.5 407.9 ± 30.8

66.7 ± 8.5t 94.2 ± 12.9 255.1 ± 47.6

547 ± 54

458 ± 30

437 ± 74

308 ± 62

26.1 ± 0.6

6.27 ± 0.17 18.8 ± 1.1

6.89 ± 0.19 12.2 ± 1.4

6.89 ± 0.17 8.8 ± 0.5

7.03 ± 0.16 8.3 ± 0.8

31.9 ± 1.9

24.4 ± 2.1

15.8 ± 2.6

11.8 ± 1.6

10.8 ± 2.2

18 (n =

6)

24 (n =

6)

LVDP. Left ventricular developed pressure; LVEDP, left ventricular end-diastolic pressure; CK, creatine kinase; ATP, adenosine triphosphate; CP, creatine phosphate. 'Functional values arc expressed at a median balloon volume of 60 1'1.

tp

< 0.05

when compared with control.

Relationship between ischemic duration and postischemic recovery. Hearts were subjected to hypothermic (10° C) global ischemia by immersion in cardioplegic solution for 6, 12, 18, or 24 hours (n = 6 per group). Relationshipbetweenstorageenvironmentand postischemic recovery. Hearts (n = 6 per group) were either suspended in moist air (inside a water-jacketed heart chamber containing a small amount of cardioplegic solution and maintained at 10° C) or were immersed in cardioplegic solution (at 10° C) for the hypothermic ischemic storage duration (6 or 12 hours). Relationshipbetween storagetemperatureand postischemic recovery. Hearts (n = 6 per group) were immersed in cardioplegic solution for 12 hours of global ischemia at various degrees of hypothermia (1.0°, 5.0°, 7.5°, 10.0°, 12.5°, 15.0°, or 20.0° C). Indices measured and expression of results. During the normothermic (37° C) preischemic period, control values for left ventricular developedpressure (LVDP) (calculated by peak systolicpressure minus end-diastolic pressure) and left ventricular end-diastolic pressure (L VEDP) were measured at different balloon loading volumes (20, 40, 60, 80, 100, and 120 JLI). After 60 minutes of reperfusion these indices were again measured, and their recovery was expressed either in absolute units of developedpressure (em H20) or as a percent of their individual preischemiccontrol values. During reperfusion the coronary effluentwas collected and analyzed for creatine kinase activity (expressed as IV /60 min/gm dry weight). Frozen tissue collected at the end of each experiment was lyophilized and the wet/ dry weight ratio was determined. The lyophilizedtissuewas also used for analysis of adenosine triphosphate (ATP) and creatine phosphate (CP) content (JLmol/gm dry weight) by a method described previously. 19 Statistics. At least six hearts were used for each condition studied, and the results were expressed as mean ± standard error of the mean. Data from all groups were tested for analysis of variance (ANOVA), and, if significance was established,

Tukey's t test was used to analyze individual groups. A value of < 0.05 was considered to indicate a significant difference.

p

Results Relationship between ischemic duration and postischemic recovery. Hearts (n = 6 per group) were subjected to a 3-minute infusion of 81. Thomas' Hospital cardioplegic solutiorr" before 6, 12, 18, or 24 hours of global ischemia (at 10° C), followed by reperfusion for 60 minutes. Function. The LVDP/volume relationship obtained at six balloon volumes after 6, 12, 18, or 24 hours of hypothermic global ischemia is shown in Fig. 1, A, and is compared with the mean control preischemic pressure/volume relationship for hearts from all groups (n = 6 per group; 24 in total). At each balloon volume there is a time-dependent deterioration of contractile function; this is shown clearly in Fig. 1, B, where the mean LVDP at a median loading volume of 60 ~l is plotted for each ischemic duration (Table II). Thus at this loading volume LVDP recovers to 84.2% ± 5.3% after 6 hours, 69.1 % ± 3.3% after 12 hours, 55.6% ± 4.4% after 18 hours, and 53.0% ± 6.8% after 24 hours. Analysis of LVEDP measurements (Fig. 2, A; see Table II) showed a threshold phenomenon such that progressive deterioration occurred after 12 and 18 hours that did not deteriorate any further after 24 hours. At a median loading volume of 60 ~l, mean LVEDP increases from the preischemic control value of all hearts (n = 24) of 3.6 ± 0.6 em H 20 (100%) to 179% ± 58%, 1458% ±

The Journal of Thoracic and Cardiovascular

Takahashi et al.

23 8

Surgery

(A)

(A)

200

6'N

200

Control

150

6h

6'N 150

~ 100

12 h

c ~

18 h 24 h

.e

100

~

50

::z:: E

a..

50

E

a. c w

o+--r----,,---.----.----,-----, o 40 ro 00 100 120 (B)

24h 18h

::z::

12h

6h Control

20

Balloon Volume (J1l)

40

ro

00

100

120

Balloon Volume (Jll) (B)

120

6' N

::z:: E

100 00

.e ro a..

c w 40 > ....J 20 O~L.J<..IC.~~~~~~=;=:::~

Fig. 1. Relationship between LVDP and (A) intraventricular balloon volume (20 to 120 ~I) after 6,12, 18, or 24 hours of global ischemia at 10.0 0 C (n = 6 per group) and (B) recovery of LVDP after 6, 12, 18, or 24 hours of global ischemia at 10.0 0 C measured at a median balloon volume of 60 ~l. Control values represent mean LVDP for all hearts (n = 24) measured after 30 minutes of aerobic control perfusion. Values are mean ± standard error of the mean. (*p < 0.05 when compared with control.

271%,2546% ± 237%, and 2616% ± 358% after 6, 12, 18, and 24 hours of ischemia, respectively. Creatine kinase leakage. Postischemic creatine kinase leakage (expressed as IU/60 min/gm dry weight) increased with increasing durations of ischemia, reaching a maximum at 18 hours. There is an apparent decrease after 24 hours of storage, however, which can be explained by the reduction in coronary effluent during Langendorff reperfusion of these hearts (see Table II). Myocardial water content. Myocardial wet/dry weight ratio was measured in fresh heart after 30 minutes

Control

12

18

24

Duration of Global Ischemia (h) Fig. 2. Relationship between LVEDP and (A)intraventricular balloon volume (20 to 120 ~l) after 6, 12, 18, or 24 hours of global ischemia at 10.0 0 C (n = 6 per group) and (B) recovery of LVEDP after 6, 12, 18, or 24 hours of global ischemia at 10.0 C (n = 6 per group) measured at a median balloon volume of 60 ~l. Control values represent mean LVEDP for all hearts (n = 24) measured after 30 minutes of aerobic control perfusion. Values are mean ± standard error of the mean. 0

of aerobic perfusion and after 60 minutes of aerobic reperfusion (at 37° C) after various durations of ischemic storage (at 10° C). In fresh hearts the ratio was 4.45 ± 0.05, which increased to 5.90 ± 0.12 after 30 minutes of aerobic perfusion. Ischemic storage for 6, 12, 18, or 24 hours followed by 60 minutes of aerobic reperfusion resulted in a progressive increase in the wet/dry weight ratio to 6.27 ± 0.17, 6.89 ± 0.19, 6.89 ± 0.17, and 7.03 ± 0.16, respectively (percent change isshown in

Volume 102 Number 2

Long-term myocardial preservation

August 1991

Fig. 3). Thus although there was a large early gain in tissue water content during aerobic perfusion, it was intensified with ischemic storage (up to 18 hours) and reperfusion. Myocardial high-energy phosphate content (ATP, CP). Myocardial content of ATP and CP (measured after 60 minutes of aerobic reperfusion) progressively declined from their preischemic control values of 26.1 ± 0.6 and 31.9 ± 1.9 ~mol/ gm dry weight, respectively, reaching a minimum value after 18 hours, with no further decrease after 24 hours of storage (Table II). Relationship between storage environment and postischemic recovery. The decreasing compliance and increasing tissue water content observed in the preceding studies suggest increasing edema as a contributory factor to the damage associated with prolonged storage of the heart. The most likely source of this fluid is that trapped in the vasculature at the time of induction of ischemia. The large volume of fluid in which the hearts are immersed, however, might also act as an external reservoir for fluid gain. To investigate this, hearts (n = 6 per group) were subjected to 6 and 12 hours ofglobal ischemia at 10° C, either immersed in 30 ml of cold cardioplegic solution (mimicking the procedure used in human heart transplantation) or suspended in moist air. Function. Pressure/volume curves were constructed before ischemia and after 60 minutes of postischemic reperfusion in all groups of hearts. Mean control preischemic values were taken from all hearts (n = 6 per group; 24 in total). Recovery of LVDP (Fig. 4, A) in hearts immersed in cardioplegic solution was slightly lower than in hearts suspended in moist air after 6 hours of global ischemia (94.9% ± 3.5%and 113.7% ± 12.4%, respectively) but slightly better after 12 hours of global ischemia (71.6% ± 2.4% and 54.2% ± 7.9%, respectively) at 10° C; these differences, however, did not reach statistical significance. Recovery of LVEDP showed a similar pattern of recovery to that of LVDP (Fig. 4, B). Creatine kinase, myocardial water content, and highenergy phosphate content. Creatine kinase (Table III) increased with increasing duration ofglobal ischemia, but there were no differences between those hearts stored in moist air or immersed in cardioplegic solution at either 6 or 12 hours, respectively. Wet/dry weight ratio was the same in both groups of hearts after 6 hours of global ischemia; however, hearts stored in cardioplegic solution had a lower wet/dry weight ratio after 12 hours of global ischemia (see Table III). These differences did not reach statistical significance. Preservation of high-energy phosphate content (see Table III) was slightly better in hearts stored in moist air than in those immersed in cardioplegic solution after 6

CP C) c: ca

oJ:

(,)

;:!!, ~ 0

'';:

~

i

~

239

200

150 100 &)

~ Q)

3=

0

Fig. 3. Relationship between wet/dry weight ratio (expressed as percent change from the in vivocondition) after 30 minutes of aerobic perfusion and 6, 12, 18, and 24 hours of global ischemia at 10.0 C and 60 minutes of reperfusion (n = 6 per group). Values are mean ± standard error of the mean. 0

hours. However, this pattern was reversed after 12 hours of global ischemic storage; these differences were not statistically significant. Relationship between storage temperature and postischemic recovery. Hearts (.11 = 6 for each condition studied) were subjected to 12 hours of global ischemia maintained at different degrees of hypothermia (1.0°, 5.0°,7.5°,10.0°,12.5°,15.0°,or 20.0° C) by immersion in cardioplegic solution. Function. The LVDP/volume relationship (measured after 60 minutes of reperfusion) for each temperature was compared with the mean control preischemic pressure/ volume relationship (Fig. 5, A) for hearts from all groups (.11 = 6 per group; 42 hearts in total). At all temperatures a significant deterioration in LVDP occurred, the pressure/volume relationship being flatter than the control. There was also a temperature-dependent decline in the absolute extent of developed pressure at any balloon volume. Taking 60 ILl as a median balloon volume, developed pressure (Fig. 5, B) recovered to around 50% of its preischemic control value at storage temperatures of 10.0° C and below, with no significant difference in recovery between hearts stored at 1.0°,5.0°, 7.5°, and 10.0° C (50.0% ± 9.3%, 60.5% ± 5.2%, 53.9% ± 3.3%, and 55.7% ± 2.2%, respectively). At storage temperatures above 10.0° C, however, a dramatic decline in recovery occurred between 10.0° and 12.5° C, and a further stepwise decrease occurred between 15.0° and 20.0° C. Similarly there was deterioration of LVEDP (Fig. 6) in a temperature-dependent manner, with a particularly

240

The Journal of Thoracic and Cardiovascular

Takahashi et al.

Surgery

(A)

(A)

!?::I

200

II Cardioplegic solution

0 N 150 E

a...

6'..... 150 E

~

100

a...

100

0

> ...J

0

> ...J

Control

::I:

::I:

~

200

Moist Air

50

50 0

0

0

1.0°C 5.0°C 7.5°C 10.0°C

~

3>

40

00

lKl

100

15.0°C 12.5°C 20.0°C

13>

Balloon Volume (Ill) (B)

150 (B)

00

0N

00

E

40

IZJ Moist Air II Cardioplegic solution

a...

100

E

~

a... c

> ...J

::I:

~

0-

::I:.....

50

0

w

> ...J

~

Temperature during Ischemia (OC)

0

Fig. 4. Relationship between recovery of (A) LVDP and (B) LVEDP after 6 and 12 hours of global ischemia at 10.0° C in hearts stored either by suspension in moist air or by immersion in cardioplegic solution measured at a median balloon volume of 60 Ill. Control values represent mean values for all hearts (n = 24) measured after 30 minutes of aerobic perfusion. Values are mean ± standard error of the mean.

rapid increase occurring between 10.00 and 12.5 0 C (Table IV). Creatine kinase leakage. Myocardial creatine kinase leakage (seeTable IV) was maximalat the highestpreservationtemperature (20.0 C); however, similarleakage was observed at all lower temperatures. Myocardial water content. Myocardial wet/dry weightratios werenot differentat any temperature studied (see Table IV). 0

Fig. 5. Relationship between LVDP and (A) intraventricular balloon volume (20 to 120 Ill) after 12 hours of global ischemia at 1.0°,5.0°,7.5°,10.0°,12.5°,15.0°,or 20.0° C (n = 6 per group) and (B) recovery of LVDP after 1.0°,5.00 , 7 . 5 ° , 10.0°, 12.5°, 15.0°, or 20.0° C(n = 6 per group) measured at a median balloon volume of 60 Ill. Control values represent mean LVDP for all hearts (n = 42) measured after 30 minutes of aerobic control perfusion. Values are mean ± standard error of the mean.

Myocardial high-energy phosphate content. High-energy phosphates (measured after 60 minutes of aerobic reperfusion) revealed a temperature-dependent deteriorationof myocardial ATP and CP content (Fig.7). Maximalvalues forbothATP and CP weremeasured inhearts storedat 1.00 C, althoughfor CP therewasnostatistically significant difference between hearts storedat 1.0 7.50, and 10.00 C, respectively. At storagetemperatures above 10.00 C there was severe depletion of both highenergy phosphates, reflecting the poor recovery of contractile function observed in these groups. 0,5.0 0

,

Volume 102 Number 2

Long-term myocardial preservation 2 4 1

August 1991

(A)

~2)

e

300

6N :I:

E

C)

20.0°C 15.0°C 12.5°C

200

.e

5.0°C 10.0°C 7.5°C 1.0°C

Q.

c w > ...J

100

O+-~:"-~=-'==~=~~ o :D 40 00 00 100 1:D

Control

Balloon Volume (J,!J)

"0

E

r2I

•

"C

AlP CP

15

~

e,

(,)

10

c..

!;;c

CO

~ co (.) 0

>-

:==

5

0

Fig. 7. Relationship between myocardial high-energy phos-

phate (ATPand CP) content and the temperature of ischemic storage after 12 hours ofglobal ischemia and60minutes ofaerobic reperfusion. Values are mean ± standard error of the mean.

(B)

200

6

N

150

:I:

E

.e Q.

100

C

w

::i

fi)

o Temperature during Ischemia (0C) Fig. 6. Relationship between LVEDP and intraventricular

balloon volume (20to 120,u1) after 12hours ofglobal ischemia at 1.0°,5.0°,7.5°,10.0°,12.5°,15.0°,or 20.0° C (n = 6 per group). Control values represent mean LVEDP for all hearts (n = 42) measured after 30 minutes of aerobic control perfusion. Values are mean ± standard errorof the mean.

Discussion Ischemic duration/recovery profile. A number of early studies on long-term preservation and heart transplantation'"21 demonstrated that adequate myocardial preservation leading to recovery of hearts was restrictedto 6 to 8 hours when usingcoldhypoxicstorage. Kondo and colleagues" stored puppy hearts in a dry hyperbaric (3 to 4 atmospheres) oxygen environment at 4 ° C for 12 hours. These hearts, when transplanted orthotopically, remained viable for up to 17 days. In another study,22 in which rat hearts were preserved with a hypothermic hyperkalemiccardioplegicsolutionfor up

to 12 hours before heterotopic transplantation in syngeneic recipients, all hearts recovered and functioned normally for up to 50 days' posttransplantation. However, ultrastructural analysisdemonstrated that, in hearts preserved for longer than 4 hours, chronic fibrosis occurred, and this was consideredto be the ultimate limiting factor for prolonged hypothermic cardiac preservation in man. More recently Swanson and colleagues-' showed that hearts arrested with a cold crystalloid cardioplegic solution and stored for 5 hours at 4 ° C recoveredbetter if they were reperfused with a bloodcardioplegicsolution than if reperfused with a hyperosmolar crystalloid cardioplegic solution. These studies demonstrate that in nonperfused hearts cold storage appears to be limited to a maximum duration of 12 hours for adequate myocardial preservation. The results from the present study confirm these observations. Goodmyocardial preservationwasobtained after 6 and 12 hours of hypothermic storage, but recovery was poor after 18 or 24 hours. Continuous perfusion at low perfusion pressures has been shown to improve postischemic function with extended durations of storage. Thus Levitsky and coworkers, 13 with use of isolated perfused dog hearts perfused continuouslywith canine plasma at a pulsatile perfusion pressure of between 15 and 25 em H 20 throughout 24-hour storage at a temperature of 8° C, demonstrated a 69% recovery of the preischemic dP /dt. Burt and Copeland, l O in a similar study with use of isolated Langendorff-perfusedrabbit hearts that were continuouslyperfusedwith a crystalloidcardioplegicsolution

The Journal of

242

Takahashi et al.

Thoracic and Cardiovascular Surgery

Table III. Effect of storage environment (moist air or cardioplegic immersion) on postischemic functional recovery. creatine kinase leakage, myocardial water content, and high-energy phosphate (ATP and CP) content in hearts subjected to either 6 or 12 hours of hypothermic (fO.O· C) global ischemia" Postischemic recovery-storage environment Ischemic duration (hr)

Control (preischemic value) (n = 24) L VDP (em H20) LVEDP (em H 20) CK leakage (IU/60 min/grn dry wt) Wet/dry wt ratio ATP content (umol/grn dry wt) CP content (umol /grn dry wt)

Moist air

6

124.6 ± 6.3 2.9 ± 0.7

Cardioplegia 12

12

6

141.7 ± 15.4 7.5 ± 0.9 134 ± 51

67.5 ± 9.8 65.0 ± 7.2 268 ± 48

6.15±0.17 20.0 ± 1.3

6.57 ± 0.09 10.7 ± 1.5

6.15 ± 0.13 18.3 ± 0.9

6.37 ± 0.15 12.3 ± 2.0

24.3 ± 3.9

14.5 ± 2.5

22.7 ± 2.7

16.2 ± 3.2

118.3 ± 4.4 8.3 ± 2.0 94 ± 25

88.8 ± 2.9 55.4 ± 9.4 323 ± 40

For explanation of terms sec Table II. "Functional values were obtained with a median balloon volume of 60 Ill.

Table IV. Effect of storage temperature on postischemic functional recovery, creatine kinase leakage, myocardial water content, and high-energy phosphate (ATP and CP) content of hearts after 12 hours' storage* Control (preischemic value) LVDP (em H 20) LVEDP (em H 20) CK leakage (lU/60 min/grn dry wt) Wet/dry wt ratio ATPeontent (umol/gm dry wt) CPcontent (umol/grn dry wt)

139.1 ± 5.0 4.5 ± 0.5

Post-ischemic recovery-storage temperature (" C)

1.0

5.0

7.5

69.6 ± 12.9 84.2 ± 7.2 75.0 ± 4.7 35.4 ± 12.5 78.3 ± 14.6 65.7 ± 6.9 343 ± 45 325 ± 13 304 ± 26

10.0

6.56 ± 0.146.46 ± 0.17 6.68 ± 0.11 7.06 ± 0.14 15.1 ± 1.8 11.3 ± 1.3 10.2 ± 1.0 8.3 ± 0.7 17.4 ± 1.4

12.6 ± 1.8

14.3 ± 2.1

12.5

15.0

20.0

33.3 ± 6.1 30.0 ± 8.8 6.7 ± 4.7 77.5 ± 3.1 70.0 ± 11.9 120.8 ± 13.9 145.0 ± 10.3 150.0 ± 20.2 280 ± 36 321 ± 43 281 ± 45 377 ± 65

12.9 ± 1.0

6.78 ± 0.40 5.5 ± 0.8

7.04 ± 0.15 6.7 ± 0.7

6.82 ± 0.08 4.1 ± 0.1

6.6 ± 1.4

6.5 ± 1.6

3.7 ± 1.4

For explanation of terms sec Table II. "Functional values were obtained at a median balloon volume of 601'1.

at a pressure of 13 mm Hg throughout a 24-hour storage period at 5° C, showed that hearts recovered to 84% of their preischemic function. In the present study recovery of myocardial function, expressed as LVDP, was approximately 50% of the control preischemic function after 24 hours of hypothermic (10.0° C) storage. Although these results appear to be similar to those of other studies in which functional recovery of hearts after a 24-hour storage period has been studied, the pressure/volume curves generated in those hearts subjected to 18 or 24 hours of cold storage were essentially flat, suggesting that contractility was poorly preserved after these storage durations. Tissue water gain. Bethencourt and Laks-" showed that dog hearts increased their weight by 29% during 24hour preservation and that it remained 28% greater than normal after reperfusion "in vitro" with blood from a

support dog. In contrast, dog hearts perfused with blood from a support dog during functional assessment, or perfused with filtered canine plasma during 24 hours of hypothermic storage, had an edema increase that remained less than 5%.13 Although the present studies allowed us to eliminate immersion as a potential source of water for cell swelling, they do highlight the need for improved osmotic and oncotic control as a component of an effective storage solution. Despite a considerable increase in wet/dry weight ratio occurring during aerobic perfusion, further increases were observed during storage or reperfusion (or both), indicating exacerbation of cell swelling. This component of myocardial injury may be readily controlled by modifying the osmotic and oncotic components of a preservation solution; however, it was not investigated in the present study. A number of studies have applied the sue-

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Long-term myocardial preservation 2 4 3

cessof long-term renal preservation with "intracellular" solutions, which were originally suggested by Collins, Bravo-Shugarman,and Terasaki-' and Sacks, Petritsch, and Kaufman." These solutions have high potassium, lowsodium,and low or zero calcium, and were advocated to reduce transmembrane cation gradients and to minimizeion exchange across the cell membrane during hypothermicpreservation. Reitz and coworkers' successfully used this type of solution to preserve dog hearts for 24 hours before transplantation and subsequent survival for up to 5.5days to graft rejection,whereas hearts stored in a modified Krebs solution were unable to support the circulation. In a similar study Toledo-Pereyra and colleagues?' were able to preservecanine hearts for up to 48 hours with the use of "intracellular" solutions with survival (determined by strength and rhythm of the heartbeat by palpitation and by electrocardiographyuntil no electricalactivity was detected) of the heterotopically transplanted hearts for up to 16 days. This study" also demonstrated that an "extracellular" solution with added colloid provided significantly more protection than Ringer's lactate solution, and these hearts had the least edema formation. An alternative was proposedby Kohno and coworkers," who obtained optimal preservation of rat hearts for 4 hours at 0 C by initially arresting the hearts with an "extracellular" solution and subsequently flushing and storing them in an "intracellular" (Collins) solution. Recently interest has developed in the University of Wisconsin solution, which was originallydeveloped for long-term preservation of the kidney, pancreas, and liverwith excellentresults. In a recent study-" University of Wisconsin solutionwas used to preservedog hearts for up to 12hours of globalischemiaand was compared with a modified Collinssolutionand Stanford solution.Hearts preserved in the University of Wisconsin solution recovered nearly normal left ventricular function after 60 minutes of reperfusion, and the hearts were able to regulate tissuewater and sodium contents to nearly normal levels. Thissolutionhas a high potassiumconcentration with low magnesiumand zero calcium; in addition it is suggested that the water content is controlled by the large impermeableanion,lactobionate (rather than chloride), and by an oncotic agent, hydroxyethyl starch. Although most cardioplegic solutions are designed to have an osmolarity in the range of 300 to 450 rrrOsm/L H 20 , little attention is paid to oncotic pressure. Increasing the colloid content of the preservation solution may allowfor improved long-term preservation,and this contention is supported by the study of Toledo-Pereyra and colleagues." The resultsofthe present study demonstrate that after excision of the heart and the establishment of aerobic 0

perfusion, a major gain in tissue water content occurred (with the wet/dry weight ratio increasing from its fresh heart value by 32.6%).This ratio became evenlarger with increasingdurations of ischemicstorage. This increase in tissue water content appears to be related to perfusion with a crystalloid solution, and has been shown to occur in other studies.I': 24 The increase in tissue water content in the present study may explain the deleterious changes in LVEDP and may also contribute to progressive myocardial failure, with the ultimate onset of irreversiblecell injury. We accept that cell swelling resulting from crystalloid perfusion solutions may be a major limitation in this type of study. An alternative, to use blood-perfused isolatedhearts, might givedifferent results. However,it is likely that this would only extend the tolerable ischemic duration achieved in the present study and that the fundamental concepts defined in the present study would remain the same. Temperature versus recovery profile. The original S1. Thomas' Hospital cardioplegic solution No.1 (STH!) has been used routinely at transplant centers such as Papworth Hospital during the past 8 years for preserving hearts during storage before transplantation.' These hearts were stored for relatively short periods with an upper limit of 4 hours. Despitethis short storage duration, however, a number of these hearts had impaired function after transplantation.v ' In a recent study from our laboratory.P the preservationcharacteristics of multidose administration of STH1 were compared with those of the more recently developed S1. Thomas' Hospital cardioplegic solutionNo.2 (STH2). It was found that postischemic recoveryof aortic flow in rat hearts after 3 hours of global ischemicarrest at 20 0 C was significantlygreater in thosehearts arrested withSTH2 (74.3% ± 6.9%) than in those arrested with STHI (18.7% ± 8.9%). All hearts arrested with STH2 had substantial recovery, whereas only six of ten hearts protected with STH1 could be converted to a functional working preparation. The major difference between the two solutions is the reduction of calcium concentration from 2.2 mmol/L (STH1) to 1.2 mmol/L (STH2), and it was suggested that calcium reduction may playa major role in the improved preservation observed with STH2. However, this study was conducted at an ischemic temperature of 20.0° C; most long-term preservationstudies have used profound hypothermia, generally around 4.0° C,4-13 and considerably enhanced preservationwouldbe expectedat lowerstorage temperatures. In this context Robinson and Harwood-? have shown that reducing the calcium in STH2 to 0.6 mmol/L can further improve the postischemicfunctional recoveryin rat hearts when stored at 20.0 C; this study has been confirmed and extended in our laboratory'? at 0

244

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Takahashi et al.

a storage temperature of7.5° C. In the present study the isolated perfused rat heart preparation! 4 has been used to investigate the long-term preservation characteristics of STH2. The results confirm that extended durations of global ischemia, up to 12 hours, were required to produce a recovery of function of approximately 50% of the preischemic function when stored at between 1.0° and 10.0° C; above 10.0° C myocardial protection was reduced, with a further reduction occurring at 20.0° C. The recovery profile relating storage temperature to postischemic functional recovery obtained in an earlier study" from these laboratories, in which a short duration (l hour) of hypothermic storage was employed, differs from the results of the present study where extended periods (12 hours) were used. In our previous study" we observed a biphasic curve over the temperature range 37.0° C to 4.0° C with an inflection between 28.0° C and 32.0° C; below 26.0° C a relatively constant levelof protection was observed, with only a small, but linear, improvement in preservation as the storage temperature was reduced to 4.0° C. Above 32.0° C there was a dramatic decline in postischemic recovery. In the present study, over the temperature range 1.0° to 20.0° C a biphasic curve was also obtained, with a sharp inflection at about 10.0° C. Above 10.0° C there was a rapid temperature-dependent decrease in postischemic functional recovery. A relatively constant degree of protection was observed from 10.0° to 1.0° C. Temperatures above 20.0° C were not investigated because survival would have been very unlikely. In comparing these results it should be noted that there were minor differences both in procedure (hearts perfused in the "Langendorff" mode in the present study versus "working" mode in the previous study) and in the formulation of the cardioplegic solution.P; however, these would seem unlikely to explain the interesting differences in results. The probable explanation lies in the different durations of storage (l hour versus 24 hours) where the nature of the developing tissue injury, particularly that relating to intracellular water gain, is more severe. On the basis of the present studies, 7.5° C appears to be the optimal temperature for long-term storage of the rat heart. Pressure/volume relationships. A feature of the pressure/volume relationships measured in the present study was that, in addition to the temperature- and timedependent depression of the curves, there was marked flattening of the ventricular function curves, the developed pressure failing to increase at larger ventricular loading volumes. This has been observed in a similar study by Burt and Copeland'? in Langendorff-perfused rabbit hearts stored for 24 hours with continuous perfusion at a

Surgery

constant perfusion pressure of 13 mm Hg, with a significant reduction of cardiac compliance and contractility demonstrated by a decrease in systolic pressure and increase in end-diastolic pressure. In the present study the stored hearts were still capable of developing a considerable systolic pressure, but there was a large increase in end-diastolic pressure when storage periods were 12 hours or greater, explaining the failure of the hearts to increase developed pressure over the range of balloon volumes studied. The increased enddiastolic pressure was probably due to the development of edema during hypothermic ischemia and reperfusion (shown by the increased wet/dry weight ratio; see Table 11). Thus the consequent decline in compliance and increase in wall stiffness may underlie the observed flattening of the postischemic ventricular function curves seen at high loading volumes.i" Concluding remarks. The present study demonstrates that extended periods of myocardial storage result in a time-dependent and temperature-dependent decrease in postischemic functional recovery, with increased enzyme leakage, tissue water gain, and reduced ATP and CP levels. The optimal preservation temperature for long-term storage appears to be 7.5° C. The nature of the storage environment (moist air or cardioplegic solution) has no significant effect on tissue water gain, viability of the preparation, or recovery. Although limited by observation in the rat heart, relatively good myocardial contractile function can be maintained with storage periods of up to 12 hours with a simple cardioplegic solution (which was developed to meet the requirements of short-term preservation, that is, less than 4 hours). In addition we do acknowledge that hearts in these studies were arrested with a crystalloid cardioplegic solution and reperfused with a crystalloid perfusate. It is conceivable that the use of blood as the perfusion and reperfusion solution may give results different from those obtained in this study. Thus effective long-term storage requires that a number of different problems be addressed, including osmotic and oncotic control, metabolite accumulation, and the way in which reperfusion is initiated. REFERENCES 1. Hearse DJ, Braimbridge MV, Jynge P. Protection of the ischemic myocardium: cardioplegia. New York: Raven Press, 1981. 2. English TA, Foreman J, Gadian DG, Pegg DE, Wheeldon D, Williams SR. Three solutions for preservation of the rabbit heart at 0° C: a comparison with phosphorus-31 nuclear magnetic resonance spectroscopy. J THORAC CAR0I0V ASC SURG 1988;96:54-61.

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3. Darracott-Cankovic S, Wheeldon D, Cory- Pearce R, Wallwork J, English TAH. Biopsy assessment of fifty hearts during transplantation. J THoRAc CARDIOVASC SURG 1987;93:95-102. 4. Kondo Y, Gradel Fa, Chaptal P-A, Meier W, Cottle HR, Kantrowitz A. Immediate and delayed orthotopic homotransplantation of the heart. J THORAC CARDIOVASC SURG 1965;50:781-9. 5. Proctor E, Parker R. Preservation of isolated heart for 72 hours. Br Med J 1968;4:296-8. 6. Freemster JA, Lillehei RC. Hypothermic-hyperbaric pulsatile perfusion for preservation of the canine heart. Transplant Proc 1969;I: 138-46. 7. Lower RR, Stofer RC, Hurley E Jr, Dong E, Cohn RB, Shumway NE. Successful homotransplantation of the canine heart after anoxic preservation for seven hours. Am J Surg 1962;104:302-6. 8. 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. 9. Wicomb WN, Cooper DKC, Barnard CN. Twenty-four hour preservation of the pig heart by a portable hypothermic perfusion system. Transplantation 1982;34:246-50. 10. Burt JM, Copeland JG. Myocardial function after preservation for 24 hours. J THORAC CARDIOVASC SURG 1986; 92:238~46.

II. Copeland JG, Jones M, Spragg R, Stinson EB. In vitro preservation of canine hearts for 24 to 28 hours followed by successful orthotopic transplantation. Surg Forum 1973; 178:687-92. 12. Proctor E, Matthews G, Archibald J. Acute orthotopic transplantation of hearts stored for 72 hours. Thorax 1971;26:99-102. 13. Levitsky S, Williams WH, Detmer DE, Mcintosh CL, Morrow AS. A functional evaluation of the preserved heart. J THORAC CARDIOVASC SURG 1970;60:625-35. 14. Langendorff O. Untersuchungen am uberlebenden Saugethierherven. Pflugers Arch 1895;61:291-332. 15. Krebs HA, Henseleit K. Untersuchungen uber die Harnstoffbildung im Tierkorper. Hoppe Seylers Z Physiol Chern 1932;210:33-66. 16. Robinson LA, Braimbridge MV, Hearse DJ. The potential hazard of particulate contamination of cardioplegic solutions. J THORAC CARDIOVASC SURG 1984;84:48-58. 17. Urdal P, Stromme JH. Effects of Ca, Mg and EGTA on creatine activity in cerebrospinal fluid. Clin Chern 1979;25:147-50. 18. Wollenberger A, Ristan 0, Schoffa G. Eine einfache Technik der extremschnellen Abkuhlung grosserer Gewebestucke. Pflugers Arch 1960;270:399-4 I2.

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19. Hearse DJ. Microbiopsy metabolite and paired flow analysis: a new rapid procedure for homogenisation, extraction and analysis of high energy phosphates and other intermediates without any errors from tissue loss. Cardiovasc Res 1984;18:384-90. 20. Ledingham SJM, Braimbridge MV, Hearse DJ. The St. Thomas' Hospital cardioplegic solution: a comparison of the efficacy of two formulations. J THORAC CARDIOVASC SURG 1987;93:240-6. 21. Toledo-Pereyra LH, Sharp HL, Condie RM, Chee M, Lillehei RC, Najarian JS. Preservation of canine hearts after warm ischemia (zero to thirty minutes) and one to two days of hypothermic storage: a comparative analysis of crystalloid and colloid solutions with different osmolarity and ion composition. J THORAC CARDIOVASC SURG 1977;74:594-603. 22. Lurie KG, Billingham ME, Masek MA, et al. Ultrastructural and functional studies on prolonged myocardial preservation in an experimental heart transplant model. J THORAC CARDIOVASC SURG 1982;84:122-9. 23. Swanson DK, Myerowitz D, Watson KM, Hegge JO, Fields BL. A comparison of blood and crystalloid cardioplegia during heart transplantation after 5 hours of cold storage. J THORAC CARDIOVASC SURG 1987;93:687-94. 24. Bethencourt DM, Laks H. Importance of edema and compliance during 24 hours of preservation of the dog heart. J THORAC CARDIOVASC SURG 1981;81:440-9. 25. Collins GM, Bravo-Shugarman M, Terasaki PI. Kidney preservation for transplantation. Lancet 1969;2:1219-22. 26. Sacks SA, Petritsch PH, Kaufman JJ. Canine kidney preservation using a new perfusate. Lancet 1973;1:1024-8. 27. Kohno H, Shiki K, Veno Y, Tokunaga K. Cold storage of the rat heart for transplantation: two types of solution required for optimal preservation. J THORAC CARDIOVASC SURG 1987;93:86-94. 28. Swanson DK, Pasaoglu I, Berkoff HA, Southard JA, Hegge JO. Improved heart preservation with UW preservation solution. J Heart Transplant 1988;7:456-67. 29. Robinson LA, Harwood DL. Lower calcium improves protection with St. Thomas' Hospital cardioplegic solution during hypothermic ischemia. J Mol Cell Cardiol 1988; 20(suppl 5):82. 30. Takahashi A, Chambers DJ, Braimbridge MV, Hearse DJ. Long term preservation of the heart: the optimal concentration of calcium in the St. Thomas' Hospital cardioplegic solution. J Mol Cell CardioI1989;21(suppl 2):361. 31. Hearse DJ, Stewart DA, Braimbridge MV. Hypothermic arrest and potassium arrest: metabolic and myocardial protection during elective cardiac arrest. Circ Res 1975; 36:481-9.