Myocardial depression after elective ischemic arrest

Myocardial depression after elective ischemic arrest Subcellular biochemistry and prevention The hemodynamic and cardiac biochemical effects of global ischemic arrest during cardiopulmonary bypass (CPB) were studied in 54 animals and compared to seven animals without ischemic arrest. Ischemic arrest alone reduced the first derivative of left ventricular force of contraction (LV dF/dt) to 52 percent of control 10 minutes after resuming function and to 64 percent after I hour of reperfusion. Cardiac output was depressed to 52 percent of control after 10 minutes of reperfusion, and to 74 percent of control after 60 minutes of reperfusion. In six animals. moderate hypothermia (26 0 C.) resulted in no protection of cardiac function from ischemic arrest. whereas profound hypothermia to 180 C. resulted in values of LV dF/dt and cardiac output nearly equivalent to the CPB control group (no arrest). A continuous infusion of a hyperkalemic hypothermic solution slightly improved the degree of protection over hypothermia alone. The sarcoplasmic reticulum (SR) isolated from hearts which had undergone 60 minutes of ischemic arrest bound significantly less calcium when the isolation was done after 10 minutes of reperfusion as well as when it was done after 60 minutes of reperfusion . The time to spontaneous release of calcium from the SR also was significantly longer. Moderate hypothermia did not result in improved SR function. whereas deep hypothermia induced by local cooling or by hypothermic potassium infusion retained SR function at normal levels. Oxidative phosphorylation oj' mitochondria isolated after 60 minutes of reperfusion was also depressed. The mitochondrial respiration rate after normothermic ischemic arrest was 155 natoms of oxygen per minutes versus 237 natoms for the hypothermic hyperkalemic group. Respiratory control index was 5.5 for the normothermic group versus 9.4 for the hypothermic group. It is concluded that hypothermia. whether effected by surface cooling or by hypothermic potassium infusion, allowed full recovery of hemodynamic and biochemical functions within hour of reperfusion.

Paul C. Gillette, M.D.,* William W. Pinsky, M.D.,* Robert M. Lewis,* Edward P. Bornet,* Jeanie M. Wood, Ph.D.,* Mark L. Entman, M.D.,* and Arnold Schwartz, Ph.D.,** Houston, Texas

T

he use of ischemic cardiac arrest during intracardiac operation predisposes to postoperative depression of myocardial function. I. 2 The prevention of this depressed cardiac function is a major problem in the field

of cardiology/cardiovascular surgery. Previous studies have shown that some cardioplegic techniques.! as well as ventricular fibrillation," are accompanied by even greater depression of hemodynamic function. Many

From the Departments of Cell Biophysics, Sections of Cardiology and Cardiovascular Sciences, Departments of Pediatrics and Medicine, Baylor College of Medicine, Texas Children's Hospital, and The Methodist Hospital, Houston, Texas.

vice, and by U.S.P.H. Grant RR-OOI88 from the General Clinical Research Branch, National Institutes of Health. Received for publication Aug. 16, 1978.

This material was developed by the Section of Myocardial Biology of the National Heart and Blood Vessel Research and Demonstration Center, Baylor College of Medicine, a grant-supported research project of the National Heart, Lung, and Blood Institute, National Institutes of Health, Grant No. HL-I7269 and Contract HV-52998.

Accepted for publication Nov. 13, 1978.

Supported in part by Grant Nos. HL-5756 and HL 13-870 from the National Institutes of Health, United States Public Health Ser-

**Present address: Department of Pharmacology, University of Cincinnati, Cincinnati, Ohio.

608

Address for reprints: Paul C. Gillette, M. D., Pediatric Cardiology, Texas Children's Hospital, 6621 Fannin, Houston, Texas 77030. *Present address: Section of Cardiovascular Sciences, Baylor College of Medicine, Houston, Texas.

0022-5223/79/040608+ I I$OJ.IO/O © 1979 The C. V. Mosby Co.

Volume

n

Elective ischemic arrest

Number 4 April, 1979

609

Table I

Group I. 2. 3. 4. 5.

CPB only Nonno.IA Mod. hypo. fA Hypo. fA Hypo. jK+ IA 6. Nonno. [K" IA 7. Hypo. 0 K+ fA

Temp. during IA (0c.)

32

26 18 18 28 18

Hyperkalemic perfusion

Temp. during measurements

No No No No

37 37 37 37 37 37 37

Yes Yes No

(OC.)

Dogs offCPB Total No. 7

20 IO

9 9 4 6

No. 7 13 6

9 9 2 I

1

Dogs with 50% CO at 10 min.

1

%

No.

100

100

60 100 100

7 6 2 9 9

50 17

I

17

65

o

%

30

20 100 100

o

Legend: Temp., Temperature. lA, Ischemic arrest. CPB, Cardiopulmonary bypass. CO, Cardiac output, Mod., Moderate. Hypo., Hypothermic. Normo., Normothermic. [K", Potassium (mEq./L.) in the perfusion solution.

methods have been proposed to protect the heart during cross-clamping of the ascending aorta. Some, such as topical or systemic hypothermia.v" have been very promising. Recently, several techniques involving infusions of various solutions into the coronary arteries during aortic cross-clamping have been proposed.v '" Experiments in rats, however, have suggested that some of these may be more harmful than helpful. 15. 16 The purpose of these experiments was to evaluate several clinically used methods of preservation and to investigate the subcellular mechanism of damage to the myocardium during ischemic arrest. An important related component of these studies was the acquisition of data on the biochemical characteristics of cardiac ischemia in general. Methods and materials Sixty-one conditioned adult dogs, 15 to 30 kilograms in weight. were used. They were anesthetized with sodium pentobarbital, 25 mg. per kilogram given intravenously. Respirations were controlled through an oral endotracheal tube with a Harvard animal respirator. Arterial blood gases, measured with a Coming Model 165 instrument, were maintained in the normal range. Rectal temperature was monitored with a Yellow Springs Instrument Co. temperature probe and thermometer. Instrumentation. A polyethylene catheter (PE 190) was advanced to the aortic arch from the femoral artery to record central aortic pressure through a Statham P23db transducer and to sample blood for blood gases. Walton-Brodie strain gauges were sutured to the free walls of the left and right ventricles. Fourteen gauge, 10 inch catheters were placed in the right ventricle from the azygos vein and in the left ventricle through the atrial appendage. In 10 animals, a Millar MicroTip catheter was placed through a stab wound in the apex of

the left ventricle. In 20 animals, a No.8 Fr., 30 cm. woven Dacron catheter was positioned in the left ventricle from the left atrial appendage and pressure was recorded with a Statham P23db transdUcer. In four dogs, both catheters were inserted and the results were compared. A Biotronix electromagnetic cuff flowmeter was placed around the ascending aorta. The flowmeter was calibrated for each experiment by allowing the dog's own blood to flow through the excised aorta into a graduated cylinder with the flowmeter in place at the end of the experiment. All measurements were recorded on a Brush eight-channel ink-jet recorder. The first derivative with respect to time of the WaltonBrodie signals and the left ventricular pressure was computed with a Biotronix differentiator and displayed simultaneously. Cardiopulmonary bypass (CPB). Each animal was given 400 units of heparin per kilogram intravenously. The left subclavian artery was cannulated with a polyethylene arterial perfusion catheter, and the superior and inferior venae cavae were cannulated through the right atrial appendage. Venous effluent, together with the right and left ventricular drainage was fed by gravity into a bubble oxygenator with a built-in heat exchanger. The arterialized blood was pumped back into the subclavian artery through an occlusive roller pump. The oxygenator was primed with donor dog blood and lactated Ringer's solution calculated to result in a hematocrit value of 26 to 28 volumes percent after CPB was begun. Blood P0 2 in the oxygenator was maintained above 100 mm. Hg and pH and PC02 were maintained at normal values by varying the flow of 95 percent oxygen and 5 percent carbon dioxide and by adding sodium bicarbonate. Total CPB was instituted. The cavae were occluded around the cannulas. The mean arterial pressure was maintained at 70 to 75 mm. Hg by controlling the rate

The Journal of

6 I 0 Gillette et al.

Thoracic and Cardiovascular Surgery

Table II. Absolute values for hemodynamic parameters before cardiopulmonary bypass (all groups) Mean ± S.D. Heart rate (beats/min.) Mean arterial pressure (mm. Hg) LV dP/dt (mm. Hg/sec.) Cardiac output (Li/min.) L VEDP (mm. Hg) RVEDP (mm. Hg)

120 ± 15 105 ± 7 1,575 ± 470 2 ± 0.3 4 ± 2 2±2

Legend: LV dP/dt, Change in left ventricular pressure and its first derivative with respect to time. LVEDP, Left ventricular end-diastolic pressure. RVEDP, Right ventricular end-diastolic pressure.

Fig. I. Schematic drawing of the experimental preparation. The arterial perfusion cannula is shown in the left subclavian artery. The electromagnetic flowmeter (EMF) is encircling the ascending aorta. The perfusion needle, which was used only in Groups 5, 6, and 7, is inserted into the aorta between the occlusion clamp and the coronary arteries. of the roller pump. Flow ranged between 75 and 100 c.c. per kilogram per minute. The left and right ventricles were vented through the pressure cannulas during CPB. Ischemic arrest. The animals were placed into six experimental groups (Table I). Group I, the control group (seven animals), was maintained on CPB for 80 minutes and then decannulated. Ten minutes after the animals' own cardiac function resumed, final recordings were made of the hemodynamic parameters measured before CPB. Group 2 (20 animals) was subjected to 60 minutes of normothermic ischemic arrest by cross-clamping the ascending aorta above the coronary arteries (Fig. I). The temperature of the myocardium was monitored after arrest by inserting a Yellow Springs needle temperature probe into the ventricle. After the 60 minutes of ischemic arrest, the clamp was removed and the heart reperfused. If ventricular fibrillation occurred, internal direct-current cardioversion was accomplished with as small a dose of energy as possible (10 to 40 watt-seconds). No drugs were used. After sinus rhythm resumed, the heart was gradually weaned from CPB and decannulated. Ten minutes (13 animals) and 60 minutes (eight animals) after decannulation, all parameters of cardiac function were remeasured. In Group 3 (ten animals), the same procedures were employed, except that the heart and blood were cooled to 26° ± 2° C. by the in-line heat exchanger before

cross-clamping. Rewarming of the blood was begun 15 minutes before removal of the clamp and was completed before beginning reperfusion. In Group 4 (nine animals), coincident with crossclamping, the exterior of the heart was immersed in 4° C. 0.9 percent sodium chloride solution in the pericardial cradle, and the interior of the left ventricle was superfused with the same solution through the left ventricular catheter. The solutions were replenished to keep the myocardial temperature at 18° ± 2° C. during the hour of ischemic arrest. The blood temperature was maintained at normal throughout. Group 5 (nine animals), Group 6 (four animals), and Group 7 (six animals) had perfusion of the aortic root through a 21 gauge needle during the hour the aorta was cross-clamped (Fig. I). In Groups 5 and 7, the perfusion solution was precooled to 4° C. The solution for Groups 5 and 6 was 5 percent dextrose in water with 15 mEg. of potassium chloride per liter; 5 mEg. per liter of sodium bicarbonate and 100,000 units of heparin per liter were also added. The final pH was 7.40 and the measured osmolarity was 310 mOsm. per liter. The P0 2 was 90 mm. Hg. In Group 5, the solution was administered at a rate sufficient to maintain the temperature of the interventricular septal myocardium at 18° ± 2° C. as measured by a needle thermistor. In Group 6, the same perfusate was used, except at room temperature. In Group 7, cold solution without potassium was infused. The effluent from the coronary sinus was allowed to mix with the blood in the oxygenator. Perfusion of the solution was stopped 5 minutes before unclamping the aorta. As soon as sinus rhythm resumed and the heart was forcefully contracting, the heart was decannulated and CPB was discontinued. The oxygenator contents were transfused into the animal until the arterial pressure and the cardiac output were judged satisfactory . Immediately before cannulation and every 10 min-

Volume 77

Elective ischemic arrest

Number 4 April,1979

6I I

Table III. Mean values of cardiac function after 60 minutes of ischemic arrest and 10 minutes of perfusion * RVEDP

Group

1. 2. 3. 4. 5. 6.

CPB only Nonno.IA Mod. hypo. IA Hypo.IA Hypo. tK+ IA Nonno. [K" IA

7 13 6 9 9 2

91 97 98 97 98 74

85 50t 58t 64t:1: 62t:1: 46t

112 52t 49t 96:j: 94:1: 41t

92 52t 42t 83:j: 73:1: 22t

96 53t 50t 84:j:

140 248 260 218 224 381'

48

280

94:1: 41

230 342

Legend: CPB, Cardiopulmonary bypass. HR, Heart rate. MAP, Mean arterial pressure. CO, Cardiac output. HR, Heart rate (beats/minute). LV dF/dt, The first derivative of left ventricular force of contraction. LVEDP, Left ventricular end-diastolic pressure. LV dP/dt, First derivative left ventricular pressure, with respect to time. RV dF/dt, First derivative of right ventricular force of contraction. RVEDP, Right ventricular end-diastolic pressure. lA, Ischemic arrest. Hypo., Hypothermic. Normo., Normothermic. [K", Potassium chloride (15 ."'Eq.IL.) in the perfusion solution. • Expressed as percent of prebypass control. tIndicates value statistically significantly different from Group I by Student's t test (p < 0.01 or greater). :j:Indicates value statistically different from Group 2 by Student's t test (p < 0.01 or greater).

utes for I hour after decannulation, the following measurements were recorded: (l) heart rate, (2) systolic, diastolic, and mean arterial pressures, (3) right and left ventricular end-diastolic pressures (R VEDP and LVEDP), (4) cardiac output, (5) force of contraction and (6) its first derivative from the left and right ventricles, and (7) left ventricular dP/dt. Biochemical methods. In Groups I, 3, and 4, and in 12 of the 20 animals in Group 2, the heart was removed 10 minutes after CPB was discontinued. In the remaining eight animals in Group 2 and in all nine of the animals in Group 5, the heart was removed 60 minutes after decannulation. The hearts were processed in a cold room for isolation of sarcoplasmic reticulum'": 19 and mitochondria." To isolate sarcoplasmic reticulum, a portion of the left ventricle was weighed, minced, and placed in three volumes of sodium bicarbonate: sodium azide aqueous solution (10:5 mM, pH 6.8). The tissue was homogenized with a Brinkman Polytron PT-35 and sarcoplasmic reticulum was isolated by differential centrifugation. 17-19 Protein was estimated by the biuret method with bovine serum albumin used as the standard. 21 Calcium binding and release parameters of isolated sarcoplasmic reticulum were assayed by the murexide method in an Aminco-Chance dual-wave length spectrophotometer.": 23 Concentrations used during calcium binding were as follows: 40 mM TRIS* maleate (pH 6.8), 10 mM magnesium chloride, 0.2 mM murexide, 100 mM potassium chloride, 40 J-LM calcium, 0.25 mM adenosine triphosphate, and 0.8 mg. per milliliter of sarcoplasmic reticulum protein. All experiments were done at 30° C. in a volume of 3.0 mI. To isolate mitochondria, a separate portion of the left ventricle was weighed and homogenized in 12 volumes *Tris (hydroxymethyl) aminomethane.

Table IV. Function of sarcoplasmic reticulum (mean ± S.E.M.)

Group

No.

I. CPB only (10 min.) 2. Nonno IA (10 min.) Nonno IA (60 min.) 3. Mod. hypo. IA (10 min.) 4. Hypo. IA (10 min.) 5. Hypo. K+ IA (60 min.)

7 12 6 6 9 9

Binding (nmolel mg. protein)

Time-to-release (min.)

45 31.6 34.5 30.0 50.0 51.0

0.87 1.80 1.65 1.85 0.85 0.88

± 4.7 ±3.7* ± 2.8* ± 4.8* ± 5.6t ± 4.3t

± 0.19 ± 0.3* ± 0.2* ± 0.38* ± 0.05t ± O.lOt

Legend: Binding, Maximal amount of bound calcium. Time-to-release, Time from initiation of the reaction until the first release of calcium was observed. The time in parenthesis indicates the duration of reperfusion before isolation. ·Significantly different from Group I by Student's t test (p < 0.01). "Significantly different from Group 2 by Student's t test (p <0.01).

of 0.18 M potassium chloride per gram, 10 mM of ethylene diamine-tetraacetate acid, and 0.5 percent bovine serum albumin (Sigma factor V), pH 7.2 to 7.4, for 3 to 4 seconds with a Brinkman Polytron PT-20 at a rheostat setting of two. Two passes through the homogenate with a motordriven Teflon pestle were then made, and the homogenate was filtered through a single layer of cheesecloth. The homogenate was centrifuged at 600 x g for 10 minutes after which the resultant cell debris and nuclear pellet were discarded. The resulting mitochondrial pellet was resuspended in approximately 0.5 C.c. of isolation medium per gram of original wet tissue. Mitochondrial respiration was measured with a vibrating platinum electrode (a Gilson Oxygraph). The Oxygraph was operated at an applied potential of -0.65 volts with the oscillator set on high. The basic assay mixture of 0.25 M sucrose, 10 mM TRIS-CI, 10 mM KP0 4 , and 5 mM substrate was present in the Oxygraph chamber. The Oxygraph was turned on and a

6 12

The Journal of Thoracic and Cardiovascular

Gillette et al.

Surgery

Table V. Mean values of cardiac function after 60 minutes of reperfusion (expressed as percent of control value) Group 2. Nanna 6. Hypo tK+

LV dF/dt

8 9

81

99

74 73

229 139

}*

204 109

}*

74 148

}*

64

96

}*

RV dF/dt

63

138

}*

Legend: Abbreviations as in Table III. "Indicates statistical significance with a p value <0.01 for a t lest between the two groups.

base line was established. The mitochondria (2 mg.) were added and allowed to equilibrate. Adenosine diphosphate (500 to 600 nmoles) was added and allowed to run until the rapid state 3 respiratory burst was completed. In the presence of maleate, mitochondrial respiration was tested with glutamate as the substrate.

Results Viability (Table I). All of the dogs from Groups I, 4, and 5 were successfully weaned from CPB. Seven of the 20 hearts which had undergone 1 hour of normothermic ischemic arrest (Group 2) could not resume any cardiac output within 15 minutes of reperfusion. Four of the 10 hearts in Group 3, two of the four in Group 6, and five of the six in Group 7 also could not be weaned from bypass. The other two animals in Group 6 had cardiac outputs less than 25 percent of control. One of these two dogs had to be put to death 20 minutes after decannulation and the other after 30 minutes because of lack of any significant cardiac output. The one animal in Group 7 which could be weaned from bypass had normal function after 1 hour of reperfusion. All others had repeated episodes of ventricular fibrillation and poor contractile function. The sixth and seventh groups of data were not subjected to statistical analysis because of the small number and because these methods of preservation obviously were not adequate. The serum potassium level was in the normal range after bypass in all groups. Myocardial temperature. Myocardial temperature during ischemic arrest in Group 2 slowly drifted down to 32° ± 2° C. even though with the heat exchanger the blood and body temperatures were maintained at 37° C. (Table I). In Group 3, the myocardial temperature was lowered to 26° ± 2° C. by cooling the blood with the heat exchanger in the arterial line before the induction of ischemic arrest. The myocardial temperature drifted up very slowly during ischemic arrest, so that at the beginning of reperfusion it was 28.5° ± 2° C. In Groups 4, 5, and 7, the myocardial temperature was maintained at 18 ± 2° C. throughout ischemic arrest. In Group 6, the myocardial temperature gradually decreased to 28° ± 4° C. during the perfusion. In all

groups, the myocardial temperature was returned to 37° ± 2° C. before the animal was weaned from CPB. Thus the control and post bypass measurements were at the same temperature. Indices of cardiovascular performance (Tables II to V). The heart rates of the animals were slightly lower after bypass in all' groups (Table III), but the difference was not statistically significant. The mean central aortic pressure 10 minutes after discontinuing bypass averaged 85 percent of prebypass values in the control animals (Table III), but this was not statistically different from prebypass values. In the other six groups, central aortic pressure was significantly depressed when compared with control (p < 0.01).

Cardiovascular function at 10 minutes of reperfusion (Table III). Three measurements were used to estimate left ventricular muscle function: (1) the force of contraction as measured by the Walton-Brodie strain gauges, (2) its first derivative (dF/dt) with respect to time, and (3) the first derivative of left ventricular pressure rise (dP/dt) with respect to time. In Groups 4 and 5 (deep hypothermia and hyperkalemia), indices were significantly closer to control function than in Groups 2 and 3, and only mean arterial pressure was significantly depressed with respect to Group 1 (Fig. 2). In Groups 5, 6, and 7, left ventricular dP/dt was not measured since it changed identically to dF /dt in the other four groups. Instead, right ventricular dF/dt was measured. Right ventricular dF /dt was significantly depressed after normothermic ischemic arrest (Group 2), but it was unchanged from control with hyperkalemic hypothermic protection (Group 5). According to these variables, when compared to Group I, postbypass left ventricular function was significantly depressed in Groups 2, 3, and 6, which had normothermic or moderately hypothermic ischemic arrest. The L VEDP and cardiac output were used as indices of cardiovascular pump function. LVEDP had been elevated by transfusion until the central aortic pressure and cardiac output were subjectively judged to be adequate as the animal was weaned from CPB. Only 140 percent of the prebypass L VEDP was required for Group I, whereas greater than 200 percent of prebypass L VEDP was required for all five groups sub-

Volume T7

Elective ischemic arrest

Number 4 April, 1979

BEFORE

AORTIC

200-

AFTER (60')

.

-

PRESSURE 10 0 (mmHg )

6I3

:.:j

0-

I

i

-

LV

-

FORCE f-

LV

-

-

dF/dl

-

-

200-

-

LV

-

PRESSURE 10 0 (mmH<;l)

• • v-.H

0-

LV

-

dp/dl

-

!

AORTIC

-

FLOW

,

40LVEOf'

(mmH<;l)

20-

0-

-

I

--

-

. --

-

-

.",aT. " JlIIlIl:lllIT1t

---

-

-

Fig. 2. Tracings of aortic pressure, left ventricularforce of contraction (LV dF ldt), left ventricular (LV) pressure and dp/dt, aortic flow. and left ventricular end-diastolic pressure (LVEDP) recorded immediately before and 10 minutes after 60 minutes of cardiopulmonary bypass without arrest. The aortic pressure is slightly lower postoperatively and the pulse pressure is wider. The parameters of left ventricular muscle function are not changed. Cardiac output and end-diastolic pressure are both slightly elevated postoperatively. jected to ischemic arrest. The resultant cardiac output was significantly depressed in Groups 2, 3, and 6 but was indistinguishable from control in Groups 4 and 5 (Table III). R VEDP was elevated to a similar degree as LVEDP in groups in which it was measured.

Function of sarcoplasmic reticulum after 10 minutes of reperfusion. Sarcoplasmic reticulum isolated from hearts which had been subjected to CPB without ischemic arrest exhibited peak binding and time-to-release within our normal values (Table IV). Sarcoplasmic reticulum from hearts subjected to normothermic ischemic arrest (Group 2) had decreased ability to bind calcium and a prolonged time-to-release (Table IV). This was also true for hearts which had undergone moderate hypothermic arrest (Group 3). There were no differences between sarcoplasmic reticulum isolated from Group 2 and Group 3 hearts (Table IV). Binding and time-to-release of sarcoplasmic reticulum from hearts which had undergone deep hypothermic ischemic arrest (Group 4) or hyperkalemic hypothermic ischemic arrest (Group 5) were indistinguishable from control (Group l) (Table IV).

Cardiovascular function at 60 minutes of reperfusion. In the groups in which cardiovascular function was measured at 60 minutes of reperfusion (Groups 2 and 5), all indices of cardiovascular performance had improved when compared to the 10 minute value (Tables III and V. Fig. 3). As a result, 60 minute values in Group 5 were not different from values in Group I. In contrast, Group 2, although improved, remained abnormal. Sarcoplasmic reticulum at 60 minutes. Sarcoplasmic reticulum fragments isolated from the hearts of the normothermic ischemic arrest group after 60 minutes of reperfusion had significantly less peak calcium binding activity than control (Table IV). Time-torelease also was significantly longer than control (Table IV). These parameters were no different from control in the hypothermic hyperkalemic arrest group. Mitochondria at 60 minutes. With glutamate as the substrate, the mitochondrial respiration rate was depressed in the normothermic ischemic arrest group after 60 minutes of reperfusion. AIl of the values for mitochondrial function were indistinguishable from control

The Journal of

6 14

Gillette

-et

al .

Thoracic and Cardiovascular Surgery

AFTER (50 ')

BEFORE

LV

FORCE

LV

dF/dt

.' ./1 "iT

.~

LV

1, "* ' 1\ 1\

dp/dt

,...... ,f\-""• . Ht--II .j4--1\ ' 1,\

,.1\

...

·11

40LVEOP (mmHg)

200-

AORTIC

FLOW

1....

Fig. 3. Tracings of cardiovascularfunction measurements (as listed in Fig. I), taken from an animal which had undergone60 minutes of normothermic ischemic arrest during cardiopulmonary bypass. There is markeddepression of all indices of left ventricular muscle function . Cardiac output is markedly depressed and end-diastolic pressure is elevated. Table VI. Mitochrondrial data QOt (nat oms

O/min ./mg.)

Group Normal Normo.

Hypo. [K

8 8 9

7.74 ± 1.3 5 .5 ± 0.9 9.4 ± 0.7

3.2 ± 0.8 2.4 ± 0.4 3.3 ± 0.3

232 ± 16 115 ± 21* 237 ± 28

Legend : Mitoch ondria values using glutamate as substrate: Norma l. Values from normal dog hearts. Hypo . [K, Hypothermic hyperkalemic co ronary perfusion group . ReI . Respiratory control index . ADP : O . The ratio of ADP added to oxgen used in oxidative phosph orylation. QO,. The respiratory rate during state 3 respiration. The values are the mean ;;; the standard error of the mean . 'p < 0 .0 I for a t test performed between the two group s.

for the mitochondria isolated from the hypothermic hyperkalemic arrest group (Table VI). Correlation of myocardial function with subcellular function. An attempt was made to test correlation between the biochemical data and the hemodynamic data. For Group 2 (ischemic arrest), the best correlation coefficient was between left ventricular dF/dt and calcium binding (r = 0.65, p = 0.1). Good correlation was obtained with the data from Group I (no ischemic arrest) using left ventricular dF/dt and calcium binding (r = 0.78, P < 0.01) . The correlation coefficient for

the data from Groups 4 and 5 was not as good : Group 4 (hypothermic arrest) left ventricular dF/dt versus calcium binding (r = 0 .45. P < 0.4); Group 5 (hypothermic hyperkalemic arrest) left ventricular dF /dt versus calcium binding (r = 0 .55. P < 0.3) . Discussion Biochemical factors in decreased myocardial function. Neither the cause of the initial decrease in contractile force in response to ischemic arrest nor the prolonged effects after reperfusion following ischemic arrest are known . In these experiments, there was an association between depression of sarcoplasmic reticulum function and prolonged decrease in cardiac contractility similar to that demonstrated in a variety of models of congestive heart failure-the Syrian cardiomyopathic hamster;" myocardial infarction owing to coronary ligation ;" the failing left ventricle of the rabbit .f" and the failing human heart.!? Statistically significant correlation was found between hemodynamic and biochemical functions only in the control group (no ischemic arrest) . There was also a strong though statistically insignificant correlation in the ischemic arrest group. The correlation was even farther

Volume 77

Elective ischemic arrest

Number 4 April,1979

BEFORE

6 I5

AFTER (60')

1 AORTIC PRESSURE (rnmHq)

LV FORCE

LV dF/dl

200LV 10 0 PRESSLRE

(mmHg )

0-

LV dp/dl

40LVEDP (mm Hg)

2 00-

AORTIC FLOW

Fig. 4. Tracing s as in Fig. 1 and 2, before and 10 minutes after 60 minutes of ischemic arrest, with the myocardium at 18° C . There is mild depression of left ventricular force and dF/dt , but left ventricular dp/dt is equal to control. Cardiac output is equal to control and end-diastolic pressure is slightly elevated.

from statistical significance in the two protected groups . Not surprisingly, it was difficult to show a statistical correlation between two separate biological functions, each of which is at best a partial measurement of multiple parameters responsible for control of the heart contraction. Also, each measurement is subject to individual experimental variables which increase the variability. Considering these problems, we feel that we have shown an association between depressed contractility and depressed function of the sarcoplasmic reticulum . The cardiac sarcoplasmic reticulum has been estimated to be able to bind and release enough calcium rapidly enough for excitation-contraction coupling of cardiac contraction and relaxation." Previous studies from this laboratory found the delayed and prolonged spontaneous release of calcium from the sarcoplasmic reticulum to be the earliest functional deficit resulting from myocardial ischemia due to coronary artery ligation. Release was abnormal at a time when calcium binding was normal. 25 In our studies both calcium binding and release were depressed after I hour of ischemic arrest and 10 and 60 minutes of reperfusion . The reasons for the differences in function of the sarcoplasmic reticulum in this study as compared to that in the occlusive ischemia study" are unknown but may be re-

lated to the fact that in the former the heart continued to perform external work whereas in the present experiments the heart stopped working within 5 minutes. This study also has shown that sarcoplasmic reticulum and mitochondria isolated from hearts which have been successfully protected hemodynamically during arrest by hypothermia or by hypothermic hyperkalemic perfusion had normal parameters of organelle function. Therefore, the preservation of mechanical characteristics of the heart appears to be paralleled biochemically by preservation of sarcoplasmic reticulum and mitochondrial function. It is possible that depression of the ability of the sarcoplasmic reticulum to release calcium results in a decreased availability of intracellular activator calcium and, thus , a decreased force of contraction. The sarcoplasmic reticulum is believed to be one of the intracellular pools from which calcium may be released during depolarization.l"?" In addition, decreased calcium transport by the sarcoplasmic reticulum might also be responsible for the overload of calcium in the mitochondria." This redistribution of calcium to a source not responsive to excitation would further depress cardiac function . Calcium loading of mitochondria also might be partially responsible for the defect in

616

The Journal of Thoracic and Cardiovascular Surgery

Gillette et al.

LVEDP

Cardiac Output

ec

100

8 '0 ~

50

~

+

[

400

f

300

e

g

u 200 '0 100

(6) (9)

(6) S)

(6) (9)

10 '

60'

10'

(6)

(9)

60'

Fig. 5. Left, Bar graph showing cardiac output in the normothermic ischemic group (shaded area) and the hypothermic hyperkalemic group (clear area) measured at 10 minutes and 60 minutes after discontinuing cardiopulmonary bypass. Right . Bar graph showing the left ventricular end-diastolic pressure (LVEDP) of the normothermic ischemia group (shaded area) and the hypothermic hyperkalemic groups (clear area) at 10 minutes and 60 minutes. All values are expressed as percent of prebypass measurements . The lines represent standard error of the mean. The numbers in parentheses represent the number of observations for each group.

mitochondrial metabolism observed. Others'" : 30 using electron microscopy have found that the 1 hour of ischemic arrest leads to disruption of the sarcoplasmic reticulum as well as to mitochondrial abnormalities . Estimation of myocardial function after ischemic arrest. Because no single method is adequate to measure myocardial "contractility," several different types of measurements were made. Since each of them changed in the same direction in each group, we believe that we were accurately estimating myocardial function. For example, in Group 2, at a time when heart rate was unchanged, afterload was reduced, and preload was increased, cardiac output was decreased from control. If myocardial performance had been unchanged, cardiac output should have been increased in the face of a decreased afterload and increased preload. The dF/dt of both ventricles and dp/dt of the left ventricle also indicated decreased myocardial performance at the same time. Protection of the myocardium from ischemia. Many methods have been used to protect the heart from ischemic arrest both clinically and experimentally, including local and systemic hypothermia, intermittent perfusion;" coronary perfusion with cold blood," Ringer's solution," ventricular fibrillation;" tetrodotoxin ," and cardioplegia with potassium citrate or acetylcholine.P Hypothermia alone has been shown re-

peatedly to be an effective method of myocardial preservation.": 6 Interventions which perfuse the coronary arteries during ischemic arrest could be either beneficial or deleterious. v '" The exact degree of local hypothermia necessary to protect the myocardium has not been determined . Our study has shown that moderate systemic hypothermia (26 0 C.) was not adequate to protect the myocardium from 1 hour of ischemic arrest. Deep, locall y induced hypothermia was found to be sufficient to give greater than 90 percent protection of myocardial function. This technique is easy to perform and has been used clinically for a number of years with good results. 35 Combined deep local hypothermia to the inner and outer walls of the left ventricle also has prevented endocardial-epicardial blood flow maldistribution in dogs with left ventricular hypertrophy. 36 Hyperkalemic hypothermic perfusion of the coronary arteries for the duration of ischemia also offered excellent protection. This technique may be less cumbersome than hypothermia alone and also stops the heart faster . Hypothermic perfusion of the coronary arteries with a solution without potassium resulted in an inability to reinstitute normal circulation, suggesting the importance of depolarization in experiments using perfusion techniques. Normothermic hyperkalemic perfusion was also ineffective. If greater than 50 percent of cardiac function is lost during CPB and ischemic arrest , the animal is unlikely to survi ve. 4 This criterion was used to construct Table I, which confirms the conclusion drawn from the whole hemodynamic data. Although great pains were taken to mimic the clinical situation in all possible experimental details, the results of this study must be interpreted with the knowledge that hemodynamic findings from dogs may not be comparable to those from human beings and that none of the hearts in this study was diseased or hypertrophied. Future studies performed on hypertrophied hearts either experimentally induced or due to naturally occurring disease in animals, would add greatly to the clinical applicability. It would also be advantageous to extend the period of observation after ischemia for days or weeks and to study other biochemical parameters of this preparation. This particular preparation of ischemia has an advantage over coronary ligation in that there is no question of collateral flow . In this way, it may be the preferable model for obtaining data on ischemia in general. REFERENCES Goldstein SM, Nelson RL, McConnell OH, Buckberg GO: Cardiac arrest after aortic cross clamping. Effectsof

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conventional vs. pharmacologic arrest on myocardial supply/demand balance. Surg Forum 26:271-273, 1975 2 Waldhausen JA, Braunwald NS, Bloodwell RD, Cornell WP, Morrow AG: Left ventricular function following cardiac arrest. J THORAC CARDIOVASC SURG 39:799-807, 1960 3 Hottenrott CE, Towers B, Kurkji HI, Maloney JY, Buckberg G: The hazard of ventricular fibrillation in the hypertrophied ventricles during cardiopulmonary bypass. J THORAC CARDIOVASC SURG 66:742-753, 1973 4 Greenberg JJ, Edmunds LH: Effect of myocardial ischemia at varying temperatures on left ventricular function and tissue oxygen tension. J THORAC CARDIOVASC SURG 42:84-91, 1961 5 Ebert PA, Greenfield U, Austen WG, Morrow AG: Experimental comparison of methods for protecting the heart during aortic occlusion. Ann Surg 155:25-32, 1962 6 Bernhard WF, Schwarz HF, Mallick NP: Profound hypothermia as an adjunct to cardiovascular surgery. J THORAC CARDIOVASC SURG 42:263-274, 1961 7 Griepp RB, Stinson EB, Shumway NE: Profound local hypothermia for myocardial protection during open-heart surgery. J THORAC CARDIOVASC SURG 66:731-741,1973 8 Hearse OJ, Stewart DA, Braimbridge MY: Hypothermic arrest and potassium arrest. Metabolic and myocardial protection during elective cardiac arrest. Circ Res 36: 471-489, 1975 9 Hearse OJ, Stewart DA, Braimbridge MY: Cellular protection during myocardial ischemia. The development and characterization of a procedure for the induction of reversible ischemic arrest. Circulation 54: 193-197, 1976 10 Paulussen F, Hubner G, Grebe D, Bretschneider HJ: Die Feinstruktur des Herzmuskels wahrend einer Ischamie mit Senkung des Energiebedarfes durch spezielle Kardioplegie. Klin Wochenschr 46: 165-169, 1968 II Reidemeister JC, Heberer G, Bretschneider HJ: Induced cardiac arrest by sodium and calcium depletion and application of procaine. Int Surg 47:536-538, 1967 12 BretschneiderHJ, HubnerG, Knoll D, LohrB, Nordbeck H, Spieckermann PG: Myocardial resistance and tolerance to ischemia. Physiological and biochemical basis. J Cardiovasc Surg 16:241-246, 1975 13 Kirsch U: Untersuchugen zum Eintritt der Totenstarre an Ischaemischen Meerschweinchenherzen in Normothermie. Arzneim Forsch 20:1071-1074, 1970 14 Kirsch U, Rodewald G, Kalmar P: Induced ischemic arrest. Clinical experience with cardioplegia in open-heart surgery. J THORAC CARDIOVASC SURG 63: 121-125, 1972 15 Hearse OJ, Steward DA, Braimbridge MY: Myocardial protection during bypass and arrest. A possible hazard with lactate-containing infusates. J THORAC CARDIOVASC SURG 72:880-883, 1976 16 Jynge P, Hearse OJ, Braimbridge MY: Myocardial protection during ischemic cardiac arrest. A possible hazard with calcium-free cardioplegic infusates. J THORAC CARDIOVASC SURG 73:848-855, 1977 17 Gillette PC, Lewis RM, Munson R, Wong N, Kaniike K,

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Schwartz A: Effects of elective ischemic arrest on myocardial performance and isolated subcellular fractions. Defect in phosphorylation of sarcoplasmic reticulum. Circulation 49,50:Suppl 3: 18, 1974 Entman ML, Snow TR, Freed D, Schwartz A: Analysis of calcium binding and relaxing system (sarcoplasmic reticulum). The use of specific inhibitors to construct a two component model for calcium binding and transport. J Bioi Chem 248:7762-7772, 1973 Harigaya S, Schwartz A: Rate of calcium binding and uptake in normal animal and failing human cardiac muscle. eire Res 25:781-794, 1969 Sordahl LA, Johnson C, Blailock ZR, Schwartz A: The mitochondrion, Methods in Pharmacology, vol I, A Schwartz, ed., New York, 1971, Appleton-CenturyCrofts, pp 247-286 Layne E: The Biuret Method. Methods in Enzymol 3: 450-453, 1957 Ohnishi T, Ebashi S: The velocity of calcium binding of isolated sarcoplasmic reticulum. J Biochem 55:599-603, 1964 Entman ML, Bornet EP, Schwartz A: Phasic components of calcium binding and release by canine cardiac relaxing system (sarcoplasmic reticulum fragments). J Mol Cell Cardiol 4: 155-169, 1972 Schwartz A, Sordahl LA, Crow CA, McCollum WB, Harigaya S, Bajusz E: Several biochemical characteristics of the cardiomyopathic Syrian hamster, Recent Advances in Studies of Cardiac Structure and Metabolism, vol I, E Bajusz, GRona, eds., Baltimore, 1972, University Park Press, pp 235-242 Schwartz A, Wood JM, Allen JC, Bornet EP, Entman ML, Goldstein MA, Sordahl LA, Suzuki M: Biochemical and morphological correlates of cardiac ischemia. I. Membrane systems. Am J Cardiol 32:46-61, 1973 Ito Y, Suko J, Chidsey CA: Intracellular calcium and myocardial contractility. Y. Calcium uptake of sarcoplasmic reticulum fractions in hypertrophied and failing rabbit hearts. J Mol Cell Cardiol 6:237-247, 1974 Solaro RJ, Briggs FN: Estimating the functional capabilities of sarcoplasmic reticulum in cardiac muscle. calcium binding. Circ Res 34:531-540, 1974 Jennings RB: Relationship of acute ischemia to functional defects and irreversibility. Circulation 53:Suppl 1:26-29, 1976 McCallister LP, Munger BL, Tyers GFO, Hughes HC: The effect of different methods of protecting the myocardium on Iysomal activation and acid phosphatase activity in the dog heart after one hour of cardiopulmonary bypass. J THORAC CARDIOVASC SURG 69:624-633, 1975 Benzing G, Stockert J, Nave E, Kaplan S: Myocardial ischaemia and cardiopulmonary bypass. Cardiovasc Res 7:186-195, 1973 Poirier RA, Guyton RA, McIntosh CL, Morrow AG: Drip retrograde coronary sinus perfusion for myocardial protection during aortic cross-clamping. J THORAC CARDIOVASC SURG 70:966-973, 1975

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32 Tyers GFO, Hughes HC, Todd GJ, Williams DR, Andrews EJ, Prophet GA, Waldhausen JA: Protection from ischemic cardiac arrest by coronary perfusion with cold Ringer's lactate solution. J THoRAc CARDIOVASC SURG 67:411-418, 1974 33 Baumann FG: The effect of normothermic anoxic arrest and ventricular fibrillation on the coronary blood flow distribution of the pig. J THoRAc CARDIOVASC SURG 69:858-859, 1975 34 Tyers GFO, Todd GJ, Niebauer 1M, Manley NJ, Waldhausen JA: Effect of intracoronary tetrodotoxin on re-

covery of the isolated working rat heart from sixty minutes of ischemia. Circulation 49,50:175-179, 1974 35 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 grafting. J THORAC CARDIOVASC SURG 70:995-1009, 1975 36 Singh H, Tector A, Flamma R, Lepley D Jr: Topical myocardial cooling in preservation of subendocardial blood flows. Circulation 52:Suppl 2:240, 1975