The relationship between myocardial temperature and recovery after experimental cardioplegic arrest

J THoRAc CARDIOVASC

SURG

84:656-666, 1982

The relationship between myocardial temperature and recovery after experimental cardioplegic arrest The aim of this study was to determine the temperature for optimal myocardial preservation during cardioplegic arrest. In isolated canine hearts perfused by a support dog, functional and metabolic recovery was measured after cardioplegic arrest using the St. Thomas' Hospital solution. The temperature range, to 37° C, was studied using a 2 hour arrest period. A 6 hour arrest period was used to enhance differences in the range 4° to 20° C. Cooling to -r C produced severe mitochondrial damage seen on electron microscopy and zero recovery offunction. Reperfusion after 2 hours of arrest at 4° or I5° C was followed by complete functional and metabolic recovery. As the temperature during arrest was raised above 20° C, recovery decreased markedly, culminating in ischemic contracture at 37° C. The severe stress of 6 hours of arrest revealed further increases in protection conferred by stepwise cooling to 4° C. In diseased hearts, long periods of ischemia are less well tolerated than in the normal hearts used in this study. Therefore, the additional protection conferred by cooling to 4° C is of clinical importance. The conclusion is that during cardioplegic arrest, provided freezing is avoided, the lower the myocardial temperature, the better is the protection against ischemia.

-r

F. L. Rosenfeldt, M.D., F.R.C.S.E. (by invitation), Melbourne, Australia Sponsored by David C. Sabiston, Jr., Durham, N. C.

Hypothermic cardioplegia has two protective components, hypothermia and cardioplegia, and their effects are additive." Comparison of the effect of hypothermia alone with that of chemical cardioplegia alone reveals that hypothermia has the more powerful protective action. This has been demonstrated in the isolated rat heart- and in the dog." In man, local cardiac hypothermia without cardioplegia was used with good results for many years," whereas chemical cardioplegia without hypothermia produced poor results" and was abandoned. Despite the use of hypothermia in cardiac operations for nearly 30 years, there is still controversy as to which temperature provides the best protection against ischemia. Some investigators have found increasing

protection with cooling to 5° C6, 7; others have concluded that 10° to 15° C8 or even 27° C9 is optimal. The aim of the present study was to measure functional and metabolic recovery of the canine heart after cardioplegic arrest at myocardial temperatures encompassing the entire range encountered in clinical operations (- 2° C to 37° C) and to determine the temperature which gave the best protection against ischemia.

Methods

Address for reprints: Dr. F. L. Rosenfeldt, Baker Medical Research Institute, Commercial Road, Prahran, Victoria, 3181, Australia.

Isolated supported heart preparation. The heart from a donor dog was excised and connected to a circuit to perfuse it in the Langendorff mode, with blood at normal temperature and pressure from a support dog. Support dog. Greyhounds of 29 kg mean weight were anesthetized with chloralose, 80 mg/kg, and mechanically ventilated. Arterial pressure, right atrial pressure, and the electrocardiogram were monitored continuously to ensure that the support dog was in a stable cardiovascular state. Blood gas analysis was done every 20 to 30 minutes and parameters maintained within physiological limits by giving sodium bicarbonate and adjusting ventilation when required. Anticoagulation was achieved with heparin, initially 300

656

0022-5223/82/110656+ 11$01. 10/0 © 1982 The C. V. Mosby Co.

From the Cardiovascular Surgical Research Unit of the Baker Medical Research Institute, Melbourne, Australia. Supported by a grant from the National Health and Medical Research Council of Australia. Read at the Sixty-second Annual Meeting of The American Association for Thoracic Surgery, Phoenix, Ariz., May 3-5,1982.

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Optimal temperature for cardioplegic arrest

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November, 1982

Blood

+

t

temperature

Pressure regulated pump

Arterial line

Heat exchanger

Pacing

pressure

Temperature

Fig. 1. Isolated supported dog heart preparation. The donor heart is perfused via the aorta (Ao) with blood from the support dog. Perfusion temperature is kept at 37° C by the heat exchanger and pressure at 100 mm Hg by the pressure-controlled roller pump. Coronary sinus blood is pumped by the right ventricle through the pulmonary artery (PA) and back to the support dog via the venous return line. L.V., Left ventricular.

units/kg and then 150 units/kg/hr. The femoral artery and vein were cannulated and connected to the perfusion circuit, which was primed with lactated Ringer's solution. Perfusion circuit. The perfusion circuit comprised an arterial line, a roller pump, a heat exchanger, aortic and pulmonary cannulas, and a venous return line (Fig. 1). Arterial pressure was measured by a pressure transducer near the aortic cannula and maintained at 100 nun Hg by a feedback system which controlled the roller pump. Blood temperature was monitored in the circuit and maintained at 37° C by the heat exchanger. The use of a ratio of 2 to 1 between the weight of the support dog and the donor dog allowed two donor hearts to be perfused simultaneously. Donor heart. The donor dog was anesthetized with chloralose (80 mg/kg), heparinized, and mechanically ventilated. Through a median sternotomy, the venae cavae were ligated and the heart excised. A purse-string suture was placed in the mitral anulus and a vent inserted in the left ventricle via the apex. A freely distensible latex balloon was passed through the mitral anulus and into the left ventricle. The balloon was attached to a stiff nylon cannula mounted on a plastic disc. This assembly was secured in the mitral anulus by the

purse-string suture. The cannula was connected to a three-way tap through which the balloon could be filled with water or left ventricular pressure measured by a Statham P23Db pressure transducer. The frequency response of this system was flat to 20 Hz. A myocardial temperature probe was inserted into the ventricular septum. Cannulas were tied into the aorta and the pulmonary artery and the heart was connected to the perfusion circuit. Local cardiac cooling was used throughout the ischemic period, averaging 9 minutes, between excision and connection to the circuit. After connection the heart was defibrillated if necessary and then allowed at least 60 minutes of nonworking perfusion before any measurements of function were made. Measurement of left ventricular function. The heart was paced at 150 beats/min by a right ventricular pacing electrode. Left ventricular pressure and other monitored variables were recorded on a Devices eight-channel pen recorder. The first derivative of left ventricular pressure (dP/dt) was generated by an active differentiator with a cutoff frequency of 90 Hz. Isovolumic left ventricular developed pressure was calculated by subtracting diastolic left ventricular pressure from systolic left ventricular pressure. The ventricular

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6 5 8 Rosenfeldt

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100 L.V. developed pressure percent control

75

50

25 2 hours

o

5

10

15

20

25

Temperature during arrest

30

35

40

(oC>

Fig. 2. Left ventricular (L. V.) developed pressure after reperfusion as a function of temperature during arrest. In the upper curve, the developed pressure after 2 hours of arrest and reperfusion in each heart is expressed as a percentage of its own prearrest control value. In the lower curve, the developed pressure after 6 hours of arrest and reperfusion in each heart is expressed as a percentage of the mean prearrest value for the 38 hearts in the 2 hour study. The points represent the mean and the bars the 95% confidence limits for the means. The stippled area represents the 95% confidence band for the prearrest control mean value for the 38 hearts in the 2 hour study.

balloon was inflated in 5 ml increments until left ventricular developed pressure and dP/dt max reached a plateau. Each incremental balloon inflation was repeated three times and the highest values of developed pressure and dP/dt max were averaged for the prearrest measure of contractility. After reperfusion, measurements of left ventricular developed pressure and dP/dt max were made at the same balloon volumes as before arrest. Changes in left ventricular compliance were detected by comparing left ventricular diastolic pressure measured before and after arrest at the maximum balloon volume. Myocardial biopsy specimens. Full-thickness biopsy specimens of the left ventricle were taken before, during, and after arrest with a high-speed drill. These 10 to 20 mg samples were immediately frozen in liquid nitrogen and were later assayed for adenosine triphosphate (ATP), creatine phosphate, and lactate using standard enzymatic methods. to. 11 Protein content was assayed using the biuret method;" All biochemical values were expressed in terms of the wet weight of the biopsy specimens. To test for effects that may have been caused by myocardial water uptake during reperfusion, biochemical results were also calculated in terms of the assayed protein content of the biopsy tissue. The differences between the temperature groups were found to be similar, regardless of whether values were expressed in terms of protein content or wet weight. For clarity, all results are given only in terms of the wet weight of the specimens.

To determine myocardial water content after reperfusion, several full thickness blocks of the left ventricle were excised, blotted, and weighed. They were then dried to constant weight and myocardial water content was calculated. Electron microscopy. In a limited ultrastructural study, material was taken for electron microscopic study from hearts in the - 2°, 4°, 15°, and 30° C groups of the 2 hour protocol. At the end of the experiment, the hearts were perfused with buffered 5% glutaraldehyde for 10 minutes. Blocks of tissue were excised from the left ventricle and placed in a fresh solution of 5% buffered glutaraldehyde for 1 hour. They were immersed in phosphate buffer overnight at 4° C, then postfixed for 1 hour in osmium tetroxide. The blocks were then dehydrated and embedded in araldite. Thin sections were cut, stained, and examined under the electron microscope. Experimental protocols. Two experimental protocols were used. In protocol I, a 2 hour arrest period was used to study the temperature range - 2° to 37° C. In protocol 2, the arrest period was lengthened to 6 hours to study the 4° to 20° C range more closely. Protocol J: Two hours of arrest. After control measurements of left ventricular function had been made on support and a biopsy specimen had been taken, cardioplegic arrest was induced. The aorta was clamped and the St. Thomas' Hospital cardioplegic solution, 15 ml/kg of donor body weight, was infused via the aortic root over 2 minutes at a pressure of 80 mm Hg. The St.

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Optimal temperature for cardioplegic arrest

Number 5 November. 1982

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125

Control

100

"'1

L.V. 75 dP/dt max. percent 50 control

6 hours

25

o

5

10 15 20 25 Temperature during arrest

\

30 (GCl

2 hours

35

40

Fig. 3. Recovery of left ventricular dP/dt max as a function of arrest temperature. The symbols are the same as in Fig. 2.

Thomas' Hospital solution No. 1 consists of Ringer's solution in which the potassium concentration has been raised to 20 mEq/L, and 16 mmoles of magnesium chloride per liter and I mmole of procaine per liter have been added. The whole heart was held at the temperature being studied by delivering the cardioplegic solution at that temperature and then immersing the whole heart in a thermostatically controlled saline bath. Hearts were arrested at each of the following temperatures: -2°,4°, 15°,20°,25°,30°, and 37°C. After 2 hours a second biopsy specimen was taken, the saline bath emptied, and the aortic cannula unclamped. Perfusion pressure was reduced to 50 mm Hg until cardiac tone returned and was then increased to 100 mm Hg. Left ventricular function was measured after 30 minutes of reperfusion and a final biopsy specimen was taken. Protocol 2: Six hours of arrest. To increase the ischemic stress and to magnify differences in protection, a second protocol was designed in which the arrest period was prolonged to 6 hours. The experimental design was changed from that of protocol 1 to avoid the deterioration in the condition of the support dog observed in our laboratory when a support dog was connected to the perfusion circuit for 8 hours or more. Therefore, in the 6 hour arrest protocol, the prearrest biopsy specimens were taken before removal of the heart from the donor dog and function was measured only after the arrest period when the heart was on support. The donor dog was anesthetized and ventilated as before, the heart exposed through a sternotomy, and a control biopsy specimen taken. The venae cavae were ligated, the aorta cross-clamped, and the cardioplegic solution infused through the aortic root as before. The

heart was excised and immediately placed in the temperature-controlled water bath. It was then instrumented as in protocol 1. Biopsy specimens were taken every 2 hours for the 6 hours of arrest. Groups of hearts were arrested at the following temperatures: 4°, 12°, and 20° C. After 6 hours, the heart was connected to the support dog. Monitoring of pressure in the left ventricular balloon was begun after 60 minutes of reperfusion and continued until recovery of function was maximal. This occurred within 90 minutes of commencing reperfusion. Measurements of left ventricular developed pressure and dP/dt max were then made as in protocol I and a final biopsy specimen was taken. Statistical analysis. Statistical comparisons were made throughout by means of the analysis of variance with multiple comparisons. 13 Results Two hours of arrest. Functional recovery. Values for left ventricular developed pressure and dP/dt max before arrest and after reperfusion in the seven groups are given in Table I. In Fig. 2 the reperfusion values in each group of hearts are expressed as a percent of the prearrest control values for the same hearts. Hearts arrested for 2 hours at 37° C developed ischemic contracture and showed virtually no recovery of function. Cooling to 30° and 25° C produced marked increases in recovery after reperfusion. In the 4°, 15°, and 20° C groups, developed pressure returned to prearrest control levels after reperfusion. In the - 2° C group, freezing of the myocardium occurred during arrest. After reperfusion, the myocardium became hemorrhagic and failed to contract. The

The Journal of Thoracic and Cardiovascular Surgery

660 Rosenfeldt

Table I. Functional and metabolic recovery after 2 hours of arrest Functional recovery Group

-2° C (n = 4)

4° C (n = 6)

15° C (n = 7)

200 C (n = 5)

25° C (n = 7)

30° C (n = 7)

37° C (n = 2)

Measurement

Developed pressure (mm Hg)

I

I

Metabolic recovery

I

dPldt max (mm Hglsec)

ATP ( pmole Igm wet wt)

4.0 ± 0.3 3.5 ± 0.3 1.0 ± 0.1 <0.001

8.1 ± 0.4 4.6 ± 0.7 1.4±0.3 <0.001

0.2 ± 0.1 1.5 ± 0.3 11.7 ± 2.2 <0.01

4.2 ± 0.1 4.2 ± 0.1 4.0 ± 0.1 0.2

6.9 ± 0.2 4.2 ± 0.4 6.7 ± 0.4 <0.001

0.8 ± 0.2 1.7 ± 0.2 0.5 ± 0.2 <0.01

3.3 ± 0.2 3.1 ± 0.1 3.6 ± 0.1 0.2

6.2 ± 0.4 2.1 ± 0.1 6.7 ± 0.2 <0.001

0.6 ± 0.2 3.1 ± 0.3 0.8 ± 0.3 <0.001

3.6 ± 0.1 3.4 ± 0.2 3.2 ± 0.2 <0.05

6.6 ± 0.5 1.4 ± 0.3 6.2 ± 0.5 <0.001

1.0 ± 0.2 6.1 ± 0.7 1.4 ± 0.2 <0.001

4.0 ± 0.1 3.5 ± 0.2 3.4±0.1 <0.001

6.7 ± 0.6 0.9 ± 0.1 7.3 ± 0.4 <0.001

1.0 ± 0.4 0.1 ± 0.6 1.3 ± 0.3 <0.001

3.4±0.1 1.9 ± 0.2 2.0 ± 0.1 <0.001

6.2 ± 0.3 0.7 ± 0.1 8.0 ± 0.3 <0.001

0.8 ± 0.4 13.7 ± 1.3 1.2 ± 0.4 <0.001

4.3 0.9 0.5

6.4 0.8 1.0

0.9 33.3 10.7

Control Arrest Reperfusion P Value

148 ± II

1,250 ± 90

0 <0.001

0 <0.001

Control Arrest Reperfusion p Value

148 ± 10

1,280 ± 140

138 ± 13 1.0

1,330 ± 140 0.7

Control Arrest Reperfusion p Value

131 ± 5

1,080 ± 100

126 ± 6 0.2

1,020 ± 80 0.2

Control Arrest Reperfusion p Value

123 ± 4

1,200 ± 110

123 ± 6 1.0

1,310 ± 150 0.1

Control Arrest Reperfusion P Value

130 ± 5'

1,430 ± 110

114 ± 6 <0.05

1,210 ± 90 <0.05

Control Arrest Reperfusion p Value

146 ± 8

1,140 ± 90

85 ± 5 <0.001

660 ± 40 <0.001

Control Arrest Reperfusion

117

1,190

4

80

Creatine phosphate (umole Igm wet wt)

Lactate (umoleIgm wet wt]

Legend: Values given are means ± SEM. The significance levels for metabolic recovery are derived from the F ratiofor differences between treatments (control,

arrest and reperfusion. ATP, Adenosine triphosphate.

pattern of recovery of dP/dt max was similar to that of developed pressure (Fig. 3). A change in left ventricular compliance after reperfusion was detectable only in the 30° and 37° C groups. In the 30° C group, there was a decrease in compliance which accounted for 25% of the decrease in developed pressure. After reperfusion in the 37° C group, the left ventricle became totally noncompliant with the development of ischemic contracture. Metabolic recovery. The results of the biopsy analyses are given in Table I and Figs. 4 and 5.

ATP. In all groups except the - 2° C group, there was no significant difference between the ATP level at the end of the arrest period and after reperfusion. In the 4° and the 15°C groups, the ATP content during arrest and after reperfusion was unchanged from prearrest levels. However, after arrest at a temperature of 20° C or above, ATP failed to return to prearrest levels. The decline in ATP below control became more marked with increasing temperature (Fig. 4). After reperfusion in the - 2° C group, levels of ATP and creatine phosphate were very low and lactate levels high.

Volume 84 Number 5 November, 1982

Optimal temperature for cardioplegic arrest

66 1

125

~

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A.T.P. percent control

75

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Fig. 4. Myocardial adenosine triphosphate (AT?) content as a function of arrest temperature. The general format is the same as in Fig. 2. The open symbols represent biopsy specimens taken at the end of the arrest period. The closed symbols represent biopsy specimens taken after reperfusion. Each value is expressed as a percentage of its own prearrest control. The points represent the means and the bars the 95% confidence band. The stippled area represents the 95% confidence band for the prearrest control mean value for all hearts. 35

30 25 Change in lactate (ll rnoVg )

20

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Fig. 5. Increase in myocardial lactate concentration above prearrest level. The symbols are the same as in Fig. 4.

CREATINE PHOSPHATE. The creatine phosphate content at the end of arrest showed a marked decline with increasing arrest temperature from 62% of control at 4° C to 37% at 15° C and 21% at 20° C. Above 20° C, creatine phosphate remained at 12% to 13% of control in all groups. Following reperfusion in the 25° and 30° C groups, creatine phosphate content exceeded control. LAcrATE. Lactate accumulation was greatest during arrest at 37° C. As the arrest temperature decreased, lactate levels decreased in exponential fashion. After reperfusion, excess lactate was washed out of the myo-

cardium in all groups except in the severely damaged - 2° and 37° C groups (Fig. 5). Water content. Myocardial water content after reperfusion ranged from 79.8% ± 0.6% in the 4° C group to 83.9% ± 0.3% in the -2° C group. There were no statistically significant differences between any of the groups. Ultrastructure. Material from the 4°, 15°, and 30° C groups appeared normal. However, material from the - 2° C group showed widespread mitochondrial damage. The mitochondria were expanded and many were ruptured. Myofibrils, however, appeared intact (Fig. 6).

The Journal of

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Thoracic and Cardiovascular Surgery

Fig. 6. Electron micrographs from "hearts in the 4° C group (left) and the - Z' C group (right). After 2 hours of ischemia at 4° C and reperfusion, normal ultrastructure is preserved. In contrast. at - 2° C gross mitochondrial swelling is seen and the mitochondria have ruptured. The myofibrils, however. are intact. The bar represents one micron.

Six hours of arrest. Functional recovery . The mean values of left ventricular developed pressure and dP/dt max after reperfusion are shown in Table II . In Figs . 2 and 3, developed pressure and dP/dt max after 6 hours of arrest are expressed as a percentage of normal prearrest values obtained from the 38 dogs in the 2 hour study. Left ventricular function was worse after 6 hours of arrest than after 2 hours, regardless of temperature. Left ventricular developed pressure was greater after 6 hours of arre st at 4° or \20 C than at 20° C (p < 0 .001). Left ventricular dP/dt max was greater after arrest at 4° or \20 C than at 20° C (p < 0 .05) . However, there was no significant difference in developed pressure or dP/dt max between the 4° C group and the 12° C group . Metabolic recovery. The results of the biopsy anal yses are given in Table II and Figs . 4, 5, and 7. ATP. In all groups there was a progressive decrease in myocardial ATP content with increasing duration of arrest. However, the lower the arrest temperature , the slower was this decrease (Fig. 7) . In none of the groups

wa s there a significant difference between the ATP level s at the end of arrest and after reperfusion. The ATP levels after arrest and after reperfusion in the 4° C group were significantly (p < 0.05) greater than the corresponding levels in the 12° C group, which in tum were significantly greater than those in the 20° C group (p < 0 .001). CREATINE PHOSPHATE. Creatine phosphate had decreased to values below 10% of control by 2 hours in the 20° C group and by 4 hours in the 12° and 4° C groups . After reperfusion , creatine phosphate levels exceeded controls in all groups (Table II). LACTATE . Lactate accumulated progressively during arrest in all groups. The lower the temperature , the less was the lactate level at the end of arrest. After reperfusion , the accumulated lactate was washed out of the myocardium in all groups (Fig . 5) . Water content. Myocardial water content after reperfusion in the three groups was 81.6% ± 1.0% at 4° C , 82 .9% ± 1.0% at 12° C , and 84 .1% ± 1.0% at 20° C (mean ± SEM). There were no significant differences between groups .

Volume 84 Number 5 November, 1982

Optimal temperature for cardioplegic arrest

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66 3

l

100

A.T.P. percent control

75 50

25

o

2

8

6

4 Time (hours)

Fig. 7. Myocardial adenosine triphosphate (A.T.P.) content as a function of time during arrest and reperfusion. The points represent the mean values and the bars the 95% confidence limits for the means. The upper stippled area represents the 95% confidence band for the mean prearrest control from all I7 hearts.

Table ll. Functional and metabolic recovery after 6 hours of arrest Functional recovery Group

Measurement

Developed pressure (mm Hg)

I (mm dPldt max Hglsec)

Metabolic recovery

ATP (umolelgm wet wt)

I (umolelgm Creatine phosphate I Lactate (umolelgm wet wt) wet wt}

4° C (n = 6)

Control Arrest Reperfusion p Value

101 ± 5 <0.001 (vs. 20° C)

890 ± 130 <0.05 (vs. 20° C)

3.8 ± 0.2 2.6 ± 0.2 2.6 ± 0.1 <0.001

4.8 ± 0.4 0.2 ± 0.1 5.8 ± 0.6 <0.001

0.8 ± 0.3 10.7 ± 1.0 0.8 ± 0.3 < o.oo I

3.9 ± 0.2 2.1 ± 0.2 2.1 ± 0.1 <0.001

5.0 ± 0.5 0.3 ± 0.1 6.6 ± 0.5 <0.001

0.7 ± 0.3 14.0 ± 1.4 0.7 ± 0.1 <0.001

3.6 ± 0.2 0.9 ± 0.3 I.I ± 0.2 <0.001

4.3 ± 0.4 0.3 ± 0.1 7.5 ± 0.6 <0.001

1.3 ± 0.3 26.4 ± 2.8 0.6 ± 0.1 <0.001

12° C (n = 6)

Control Arrest Reperfusion p Value

95 ± 5 >0.5 (vs. 4° C)

790 ± 90 >0.5 (vs. 4° C)

20° C (n = 5)

Control Arrest Reperfusion p Value

56 ± 5 <0.001 (vs. 12° C)

460 ± 90 >0.05 (vs. 12° C)

Legend: All significance levels for metabolic recovery were derived from the F ratio for differences hetween the three treatments (control, arrest, and reperfusion). ATP. Adenosine triphosphate.

Discussion

The advantage of the isolated supported canine heart model over the commonly used isolated rat heart preparation is that it is perfused with blood with the normal background of substrates and hormones. Also, this model avoids the blood trauma and deterioration in organ systems caused by prolonged extracorporeal cir-

culation and permits more precise control of myocardial temperature in all areas of the heart than is possible with the heart in situ. The results of the present study are entirely consistent with the concept that during cardioplegic arrest, provided freezing is avoided, the lower the myocardial temperature, the better is the protection against isch-

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emia. The 2 hour arrest protocol showed an improvement in protection with each increment in hyopothermia down to 20° C. Below this temperature, full recovery was seen after reperfusion. When the ischemic stress was increased by prolonging arrest to 6 hours, cooling from 20° to 4° C was also shown to bring about an increase in protection. Improvements in protection were seen in the measurements of recovery of contractile function after reperfusion, assessed by left ventricular developed pressure and dP/dt, and also in the metabolic response to ischemia and reperfusion, assessed by changes in ATP, creatine phosphate, and lactate. No detrimental effects of cooling to 4° C were seen in this study. However, when the temperature during 2 hours of arrest was - 2° C, the temperature of saline slush, severe damage to the heart was shown by all functional and metabolic measurements. This evidence correlated with gross mitochondrial damage seen under the electron microscope. No such damage was seen in the 4° C or other groups. A consistent finding in this study was that although reperfusion brought about a return of contractile activity, there was not an accompanying rise in the ATP level above that observed at the end of arrest. This may be largely explained by the breakdown during ischemia of ATP to adenosine and finally to inosine, xanthine, and hypoxanthine. During reperfusion, these ATP precursors are washed out of the myocardium. 14 Thus ATP regeneration after reperfusion is limited by the availability of adenosine and its precursors. Creatine phosphate is rapidly regenerated after reperfusion, but it cannot be used to resynthesize ATP in the absence of the necessary building blocks. Thus ATP regeneration would be expected to be slow and not appreciable in the 30 to 60 minute reperfusion period in the present study. There is vast literature concerning the effects of hypothermia on the body and on the heart. In general, an inverse relationship has been found between temperature on the one hand and tissue metabolic rate and oxygen consumption on the other. However, in applying the results of basic scientific studies to clinical hypothermic cardioplegia, one must concentrate on studies carried out in clinically relevant models. Tyers and colleagues" used the isolated working rat heart preparation to study the effects of various grades of hypothermia induced by infusion of a noncardioplegic electrolyte solution. The infusion was given at various temperatures and the heart then was allowed to drift toward room temperature. They found that, after reperfusion, functional and metabolic recovery was better in hearts cooled by perfusion at 10° and 15° C than at 4° C. At the end of ischemia, although levels of glyco-

Thoracic and Cardiovascular Surgery

gen and high-energy phosphates were highest in hearts perfused at 4° C, following reperfusion these hearts showed metabolic deterioration evidenced by falling levels of these compounds. In contrast, hearts initially infused at 10°and 15°C showed the best metabolic and functional recovery after reperfusion. These investigators suggested that the inadequate metabolic recovery of the 4° C hearts was due to permanent damage possibly caused by temporary solidification of membrane lipids and damage to membrane ATPase or both. Flaherty and associates" studied the effects of noncardioplegic ischemia at various temperatures in the isolated cat heart. They concluded that myocardial preservation was optimal when hearts were cooled to 27° C during ischemia. Further cooling of the heart to 20° or 10° C, while reducing the accumulation of carbon dioxide in the myocardium during arrest, resulted in no additional recovery of prearrest ventricular function. Temperatures below 10° C were not studied. In marked contrast to these results are the findings of Hearse," Harlan, 7 and their colleagues. Both of these groups used isolated working rat hearts arrested at various temperatures by a cardioplegic solution. Both groups found the greatest protection against ischemia at a myocardial temperature of 4° C. The findings of the latter two studies accord with those of the present study. How can the discrepancy between these results and those of Tyers and Flaherty be explained? The answer almost certainly is that in the studies by Hearse, Harlan, and my colleagues, a cardioplegic infusion was used at the onset of ischemia, whereas in the studies of Tyers and Flaherty, no cardioplegic solution was used. When the heart is arrested by hypothermia alone, the onset of arrest is preceded by ventricular fibrillation or by a period of bradycardia with sustained hypertonic energy-wasting contractions. This sequence of events leaves the heart with depleted energy stores at the onset of ischemia. In contrast, in hypothermic cardioplegic arrest under optimal conditions, the heart is arrested within a few beats of occluding the aorta and thus has enough stored high-energy phosphate to satisfy the small cellular energy requirements during arrest. A study by Shragge, Digerness, and Blackstone'! also supports this view. These investigators perfused isolated rat hearts with an oxygenated noncardioplegic electrolyte solution for 20 minutes at temperatures between 0.5° and 20° C. Cardiac function and highenergy phosphtes were measured at 37° C, before and after the period of hypothermic perfusion. Postrecovery function in hearts exposed to hypothermic perfusion was not significantly different from that observed in hearts kept at 37° C. When exposure time to perfusion

Volume 84

Optimal temperature for cardioplegic arrest

Number 5 November, 1982

at 0.5° C was extended to 2 hours, cardiac function still returned to the same level as that of control hearts maintained at 37° C and ATP and glycogen levels were higher than those in the control group. Thus the hypothermia- induced permanent damage to membranes, lipids, and ATPase postulated by Tyers and Flaherty was not encountered in Shragge's study even during 2 hours' exposure to a temperature of 0.5° C. However, since the hearts were perfused continuously during hypothermia with an oxygenated perfusate, hypertonic contractions or ventricular fibrillation would not deplete vital energy stores, as may have occurred in the studies of Tyers and Flaherty. The hearts in this study were from normal, healthy dogs in which cardioplegia was induced and hypothermia maintained under carefully controlled conditions. In clinical operations, although ischemic intervals are usually shorter, conditions are rarely as favorable. Hearts with ventricular hypertrophy have been shown to have impaired mitochondrial respiration, low levels of ATP, and poor subendocardial perfusion.!" As a result, they are more sensitive to the effects of ischemia than are normal hearts."? Thus it would be unjustifiable to assume on the basis of this present study that, under clinical conditions, 2 hours of arrest at 20° C would be followed by near normal recovery. In hearts with diseased coronary arteries, distribution of the cardioplegic solution is not uniform and myocardial cooling is patchy." Thus, because of various pitfalls in technique;" the intended degree of hypothermia may not be achieved in the operating room, and tolerance of the heart to ischemia may be impaired. It is imperative to have a generous safety margin included in the temperature X time calculation of the tolerable degree of ischemia. The use of cold slush runs the risk of causing freezing injury to the myocardium. Once the benign and beneficial nature of cardioplegic arrest at temperatures down to 4° C has been accepted, high-efficiency techniques of topical cooling which carry no risk of freezing'" may be used with confidence. Cooling the outside and where possible the interior of the heart chambers to 4° to 10° C helps to ensure that all regions of the myocardium are well below 20° C. The conclusion from this study is that during cardioplegic arrest, provided freezing is avoided, the lower the myocardial temperature the greater is the protection against ischemia. I wish to thank the following for advice and assistance: Mr. G. Stirling, Dr. A. Bobik, Dr. G. Campbell, and Professor J. Ludbrook. Statistical advice was given by J. Gipps, B.Sc.

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(statistics). Technical help was provided by Janet Ness, Jenny Griffiths, Jan Dixon, and Karen Kerr.

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REFERENCES Rosenfeldt FL, Hearse DJ, Cankovic- Darracott S, Braimbridge MV: The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the dog. J THoRAc CARDIOVASC SURG 79:29-38, 1980 Hearse DJ, Stewart DA, Braimbridge MV: Hypothermic arrest and potassium arrest. Metabolic and myocardial protection during elective cardiac arrest. Circ Res 36: 481-489, 1975 Kugelberg J, Hagerdal M, Carlsson C: Myocardial protection during heart surgery. An experimental evaluation of normothermic and hypothermic cardioplegia. Scand J Thorac Cardiovasc Surg 13:47-52, 1979 Griepp RB, Stinson EB, Oyer PE, Copeland JG, Shumway NE: The superiority of aortic cross-clamping with profound local hypothermia for myocardial protection during aorta-coronary bypass qrafting. J THoRAc CARD10VASC SURG 70:995-1009, 1975 Kirsch U, Rodewald F, Kalmar P: Induced ischemic arrest. J THORAC CARDIOVASC SURG 63:121-130, 1972 Hearse DJ, Stewart DA, Braimbridge MV: Cellular protection during myocardial ischemia. The development and characterization of a procedure for the induction of reversible ischemic arrest. Circulation 54: 193-202, 1976 Harlan BJ, Ross D, Macmanus Q, Knight R, Luber J, Starr A: Cardioplegic solutions for myocardial preservation. Analysis of hypothermic arrest, potassium arrest and procaine arrest. Circulation 58:Suppl 1:114-118, 1978. Tyers GFO, Williams EH, Hughes HC, Todd GJ: Effect of perfusion temperature on myocardial protection from ischemia. J THoRAc CARDIOVASC SURG 73:766-771, 1977 Flaherty IT, Schaff HV, Goldman RA, Gott VL: Metabolic and functional effects of progressive degrees of hypothermia during global ischemia. Am J Physiol 236:H839-H845, 1979 Lamprecht W, Stein P, Heinz F, Weisser H: Creatine phosphate. Determination with creatine kinase, hexokinase and glucose-S-phosphate dehydrogenase, Methods of Enzymatic Analysis, HU Bergmeyer, ed., New York, 1974, Academic Press, Inc., pp 1777-1781 Gutmann I, Wahlefeld AW: L-( + )-Lactate. Determination with lactate dehydrogenase and NAD, Methods of Enzymatic Analysis, HU Bergmeyer, ed., New York, 1974, Academic Press, Inc., pp 1464-1468 Albro PW: Determination of protein in preparations of microsomes. Anal Biochem 64:485-493, 1975 Snedecor GW, Cochran WG: Statistical Methods, ed 3, Ames, Iowa, 1967, Iowa State University Press, pp 258338 Foker JE, Einzig S, Wang T: Adenosine metabolism and myocardial preservation. Consequences of adenosine catabolism on myocardial high-energy compounds and tis-

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sue blood flow. J THORAC CARDIOV ASC SURG 80:506516, 1980 Shragge BW, Digerness SB, Blackstone EH: Complete recovery of the heart following exposure to profound hypothermia. J THoRAc CARDIOVASC SURG 81:455-458, 1981 Attarian DE, Jones RN, Currie WD, Hill RC, Sink JD, Olsen CO, Chitwood WR, Wechsler AS: Characteristics of chronic left ventricular hypertrophy induced by subcoronary valvular aortic stenosis. I. Myocardial blood flow and metabolism. J THORAC CARDIOVASC SURG 81:382-388, 1981 Attarian DE, Jones RN, Currie WD, Hill RC, Sink JD, Olsen CO, Chitwood WR, Wechsler AS: Characteristics of chronic left ventricular hypertrophy induced by subcoronary valvular aortic stenosis. II. Response to ischemia. J THoRAc CARDIOVASC SURG 81:389-395, 1981 Landymore RW, Tice 0, Trehan N, Spencer F: Importance of topical hypothermia to ensure uniform myocardial cooling during coronary artery bypass. J THoRAc CARDIOVASC SURG 82:832-836, 1981 Rosenfeldt FL: Hypothermic preservation techniquespitfalls, The Handbook of Clinical Cardioplegia, RM Engelman, S Levitski, eds., New York, 1982, Futura Publishing Co Rosenfeldt FL, Fambiatos A, Pastoriza-Pinol J, Stirling OR: A recirculating cooling system for improved topical cardiac hypothermia. Ann Thorac Surg 32:40 1-405, 1981

Discussion DR. HENDRICK B. BARNER St. Louis. Mo.

Dr. Rosenfeldts group has performed an excellent study confirming in the dog the importance of profound myocardial cooling in cardioplegic myocardial preservation, which was demonstrated previously by David Hearse and Mark Braimbridge and their associates at St. Thomas' Hospital using the isolated perfused rat heart model. Our studies of cold blood potassium cardioplegia have all utilized a myocardial temperature of 5° to 10° C, and clinically we have attempted to attain this degree of myocardial cooling but have usually fallen short by 5° C or so. In the abstract, where ATP values are recorded, it is puzzling to me that the reperfusion values for ATP were at the same level as the last ischemic values, because the phenomenon of postischemic adenosine washout resulting in lower ATP values after reperfusion of ischemic myocardium is now a well recognized and uniform phenomenon. We have tried to reverse this occurrence by pretreatment with di-

Thoracic and Cardiovascular Surgery

pyridamole, which is alleged to block adenosine transfer across the cell membrane, but have been unsuccessful in doing so. I would like to ask Dr. Rosenfeldt his thoughts on his failure to see a postischemic reperfusion decline in ATP. DR. ROBERT J. ELLIS San Francisco. Calif.

A few years ago at this meeting I presented data to indicate that perfusion hypothermia was as good with normal potassium concentration as with higher potassium concentration. I would like the author to comment on whether normal or lower concentrations of potassium were used. Also, was there any differences in the high-energy phosphate stores with lowered potassium concentrations? In studies of 100 patients undergoing myocardial biopsy, we found no difference in the level of high-energy phosphates when 5 mEq/L of potassium was compared to 20 mEq/L at cardioplegic arrest times of up to 100 minutes. To shed light on Dr. Barner's questions regarding the decreased levels of ATP after reperfusion, we studied myocardial biopsy specimens after 30 minutes of reperfusion in 15 patients and demonstrated no fall in ATP from the control specimen or the specimen taken at the end of cardioplegic arrest. We are convinced that the reperfusion injury marked by severe decreases in ATP after reperfusion seen in dogs is not present in patients with coronary artery disease. DR. R 0 SEN FE LOT (Closing) I would like to thank Dr. Barner and Dr. Ellis for their comments. Dr. Barner asked a question but also helped me answer it. I believe that the failure of ATP to return to control levels in the period of reperfusion that we allowed, 30 to 60 minutes, is probably explained by the washout from the myocardium of adenosine and its breakdown products xanthine, hypoxanthine, and inosine. This process has been very ably demonstrated by Dr. Robert Anderson's group in Minneapolis. I believe that had we studied these hearts over a number of hours, perhaps 6 or 8 hours of reperfusion, we would have seen a rise in ATP. However, as the building blocks were not there in the short term, it was not possible for the heart to resynthesize ATP. Regarding the potassium concentration, Dr. Ellis, I am sure that one could use a lower concentration of potassium at very low temperatures, 4° C, for example, and still maintain arrest. In clinical practice, it is not always possible to attain reliably temperatures below 10° C throughout the myocardium. Therefore, the extra bonus of protection provided by the higher potassium concentration is important. However, I do not believe that potassium concentration should be excessively high, particularly during reinfusion, which probably means not above 20 mEq/L.