The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the rat

J THoRAc

CARDIOVASC SURG

79:39-43, 1980

The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the rat In a rat heart model of cardiopulmonary bypass and ischemic cardiac arrest the potential additive protective effects of hypothermia and chemical cardioplegia have been investigated. Isolated rat hearts were subjected to a 2 minute period of coronary infusion with a cardioplegic or a noncardioplegic solution immediately before and also at the midpoint of a 2 hour period of hypothermic (20 0 C) ischemic cardiac arrest. In the hypothermia plus cardioplegia group postischemic aortic flow recovered to more than 50% of its preischemic control value. myocardial energy phosphate content returned to near preischemic control levels. and creatine kinase leakage was moderate. By contrast, in the hypothermia alone group (coronary infusion with non cardioplegic solution) the postischemic functional recovery was less than 30% of its preischemic control value, cellular high-energy phosphate content was considerably reduced, and creatine kinase leakage was more than twice that observed in the hypothermia plus cardioplegia group. In addition to illustrating the additive nature and powerful protective properties of hypothermia and cardioplegia these studies serve to illustrate the utility of the isolated rat heart model for the primary assessment of procedures designed to protect the myocardium during ischemic cardiac arrest. The results and conclusions derived from this study were quantitatively and qualitatively similar to those obtained in a parallel study in the dog. I

D. J. Hearse, Ph.D., D. A. Stewart, M.Sc., and M. V. Braimbridge, F.R.C.S., London, England

A

s discussed in the preceding paper, 1 there is a large amount of clinical and experimental evidence"?" that both hypothermia and chemical cardioplegia afford effective protection during ischemic cardiac arrest, but whether or to what extent the two procedures are additive is the topic of current active investigation. I, 11-15 In the preceding study 1 the protective effects of carefully controlled hypothermia (20 C) were compared with those of hypothermia (20 C) plus chemical cardioplegia. The study was carried out in the dog subjected to 120 minutes of ischemic arrest. Relative patterns of tissue protection were assessed with the use of functional, biochemical, and cytochemical indices and the 0

0

From The Rayne Institute, St. Thomas' Hospital, London SEI, England. This work was supported by grants from the British Heart Foundation and the Wellcome Trust. Received for publication June 17, 1978. Accepted for publication Nov. II, 1978. Address for reprints: Dr. D. J. Hearse, Myocardial Metabolism Research Laboratories, The Rayne Institute, St. Thomas' Hospital, London SEI, England.

results indicated that chemical cardioplegia afforded considerable protection to the ischemic heart, which was additive to that conferred by hypothermia. By necessity, the assessment of any intervention designed for human use must be carried out in an experimental model and must therefore be restricted by the limitations of that model. Although an in situ dog heart preparation is generally regarded as an acceptable model for the human heart, it does present a number of problems, not the least of which are expense, poor tolerance to extended periods of cardiopulmonary bypass, and an inability to be studied quickly and in large numbers. For these and other reasons we have in the past adopted an isolated rat heart model of cardiopulmonary bypass and ischemic cardiac arrest. 15-21 We believe this preparation to be a good biochemical model for the human heart which offers many advantages including economy, speed of preparation, reproducibility, and the ability to handle large numbers. In an attempt to illustrate the value of this preparation and also to provide further evidence that the protective effects of hypothermia and chemical cardiople-

0022-5223/801010039+05$00.5010 © 1980 The C. V. Mosby Co.

39

40

The Journal of Thoracic and Cardiovascular Surgery

Hearse, Stewart, Braimbridge

Table I. Solution infused in cardioplegic group Components

Grams lliter

mmoles lliter

NaCl KCI NaHC03 MgSO.· 7H2O MgCI2' 6H2O KH 2PO. CaCl 2 ATP . Na23H20

4.20 1.10 2.10 0.30 3.05 0.16 0.18 6.05

72.0 14.8 25.0 1.2 15.0 1.2 1.2 10.0

gia are additive, the preceding study! has been repeated using the isolated rat heart.

Materials and methods Hearts. Hearts were obtained from male rats (280 to 320 gm of body weight) of the Wistar strain. The experimental model. The isolated perfused working rat heart model, which has already been described in detail, 10. !7is a left heart preparation in which oxygenated perfusion medium (at 37° C) enters the cannulated left atrium at a pressure of 20 em H 20 and is passed to the ventricle, from which it is spontaneously ejected (electrical pacing was not used in this study) via an aortic cannula, against a hydrostatic pressure of 100 cm H 20. Coronary effluent can be sampled for analysis or pooled and recirculated with the aortic outflow. During this working period various indices of cardiac function may be measured. Total cardiopulmonary bypass with maintained coronary perfusion may be simulated by clamping the left atrial cannula and introducing perfusion fluid at 37° C into the aorta from a reservoir located 100 ern above the heart. This preparation, which is essentially that described by Langendorff'," will continue to beat but does not perform any external work. Ischemic cardiac arrest may be induced in this preparation by clamping the aortic cannula. Short periods of preischemic coronary infusion (at 37° C or any degree of hypothermia) of cardioplegic or other solutions may be achieved by use of a reservoir (located 60 cm above the heart) attached to a side arm of the aortic cannula. During the period of ischemic arrest the heart is maintained in a sealed water-jacketed container which may be maintained at 37° C or any degree of hypothermia. Secondary infusions of cardioplegic or other solutions during the ischemic period may be achieved via the side arm attached to the aortic cannula. The experimental time course. Immediately after excision of the heart the aorta was cannulated and Langendorff perfusion was initiated for a 5 minute pe-

riod, during which time the left atrium was cannulated. During this and subsequent periods the perfusion fluid was bicarbonate buffer, 10. 11 pH 7.4, containing 11.1 mmoles of glucose per liter, gassed with 95% oxygen and 5% carbon dioxde. The heart was converted to a working preparation by terminating the retrograde aortic perfusion and initiating left atrial perfusion. During a IS minute period control values for aortic and coronary flow rates, peak aortic pressure, and heart rate were recorded. At the end of this control period total cardiopulmonary bypass with maintained coronary perfusion was simulated by clamping the atrial cannula and converting the preparation to the Langendorff perfusion mode. After a further 5 minutes the aortic cannula was clamped and coronary infusion at 20° C of similar volumes of cardioplegic or a noncardioplegic solution was initiated and maintained for a 2 minute period. The coronary infusion was then terminated and the hearts were maintained in an ischemic state at 20° C for 120 minutes. After 60 minutes of this ischemic period had elapsed a secondary infusion of cardioplegic or noncardioplegic solution at 20° C was initiated and maintained for a 2 minute period. At the end of the 120 minute period of ischemic arrest the heart were reperfused at 37° C for 30 minutes in the Langendorff mode. After this period of simulated total cardiopulmonary bypass with maintained coronary perfusion the preparations were converted to the working mode and the recovery was monitored over a 30 minute period. Functional assessment. The recovery of aortic flow during the final 30 minute working period was expressed as a percent of the preischemic control value. In this way functional recovery could be related to the duration of ischemic arrest, the degree of hypothermia, and the nature of the coronary infusate. Biochemical assessment. In a parallel series of studies hearts were freeze clamped'" and taken for the determination'": 27 of adenosine triphosphate (ATP) and creatine phosphate (CP). Freeze clamping was at the end of the 15 minute preischemic control period, after 110 minutes of ischemic arrest, and at the end of the final working heart recovery period. In all instances the tissue content was expressed as micro moles per gram of dry weight. Enzymatic assessment. We have previously shown'? in this rat heart model that the activity of creatine kinase in the coronary effluent during the phase of postischemic reperfusion is a good indicator of tissue damage and functional recovery. In all experiments therefore the coronary effluent during the immediate postischemic reperfusion period (30 minutes) was collected in 5 minute batches into a container cooled to 4° C.

Volume 79 Number 1

January, 1980

Additive effects of cardioplegia and hypothermia

4I

80

60

> C<

....

o>

u .... C< I-

~ u

C< .... a.

40

20

TIME (min I

Fig. 1. Postischemic recovery of aortic flow. The recovery is expressed as a percent of the preischemic control value. Circles. Hypothermia plus cardioplegia group. Squares. Hypothermia alone group. There were 10 hearts in each group and the SEM is indicated. These fractions were analyzed'" for total creatine kinase leakage (milli-international units of creatine kinase released per minute). Infusion solutions. Hearts were subjected to 2 minute periods of preischemic and midischemic coronary infusion. In the control group the noncardioplegic solutions was unmodified Ringer's solution (NaCl 147.13 mmoles/L, KCl 4.20 mmoles/L, CaCl 2 2.25 rnrnoles/L). In the cardioplegia group the solution infused was as shown in Table I. Results Functional assessment. Fig. I shows the postischernie recovery of aortic flow rate expressed as a percent of the preischemic control. The hypothermia plus cardioplegia group (n = 10) recovered at a greater rate and to a greater extent (51. 3% ± 8.1 % after 30 minutes) than the hypothermia alone group (n = 10) 29.3% ± 7.9% after 30 minutes. Biochemical assessment. Table II shows the values for the tissue content of ATP and CP at various times in the experimental period. After 110 minutes of ischemic arrest ATP and CP had fallen to similar low levels in both experimental groups. At the end of the recovery period in both groups ATP and CP had recovered con-

siderably but the recovery of each metabolite was greater in the hypothermia plus cardioplegia group than in the hypothermia alone group. This observation would be consistent with the superior functional recovery observed for this group and is also in full agreement with the results observed in the dog heart in the preceding paper. 1 Enzymatic assessment. Fig. 2 shows the creatine kinase leakage. Over each collection period the hypothermia along group consistently released significantly more creatine kinase than the hypothermia plus cardioplegia group. Over the entire 30 minute period the total enzyme leakage was over 50% greater in the hypothermia group (18,900 ± 2,311 mID) than in the hypothermia plus cardioplegia group (12,330 ± 1,541 mID). These results would further confirm the additive protective properties of hypothermia and cardioplegia. Discussion Two points emerge from this study, The first is that functional, biochemical, and enzymatic assessments indicate that hypothermia and cardioplegia are additive, confirming the finding of the preceding paper. 1 Dnder the conditions of this study, the use of uniform hypothermia of 20° C resulted in a 30% recovery of

42

The Journal of Thoracic and Cardiovascular Surgery

Hearse, Stewart, Braimbridge

1000

800

...,; ~

""

u

:::i

600 400

--= ....l ....l

:E

200

0

0 -5

5 -

10

10- 15

15 - 20

20 - 25

25-30 MINS

POST ISCHEMIC COLLECTION PERIODS

D

HYPOTHERMIA PLUS CARDIOPLEGIA HYPOTHERMIA ALONE

Fig. 2. Postischemic creatinekinaserelease. The histogram showsthe creatinekinasereleased (mIU/min) during each 5 minute collection period during the first 30 minute period of postischemic reperfusion. Each group is the mean of 13 hearts; the SEM is indicated. Table II. Tissue metabolite values* Ischemic period

Control period

Group

Hypothermia alone Hypothermia plus

Beperfusion period

ATP (mmoles/gm dry wt)

CP (mmoles/gm dry wt)

ATP (mmoles/gm dry wt)

CP (mmoles/gm dry wt)

ATP (mmoles/gm dry wt)

CP (mmoles/gm dry wt)

17.3 ± 1.3 17.3 ± 1.3

15.4 ± 1.5 15.4 ± 1.5

4.2 ± 0.3 3.8 ± 0.8

2.6 ± 0.2 3.0 ± 0.4

9.0 ± 0.5 12.1 ± 0.9

14.4 ± 1.1 16.3 ± 0.9

cardioplegia *ATP and CP content were determined at the end of the preischemic control period. after tID minutes of the ischemic period and at the end of the working reperfusion period. Six hearts were assayed for each group at each time point and the SEM is indicated in the table.

cardiac function after a 2 hour period of ischemic arrest. In previous studies'" we have shown that the isolated rat heart recovers only 12% of its preischemic aortic flow after 30 minutes of normothermic ischemic arrest and thus the hypothermia used in the present study clearly afforded a high degree of protection to the ischemic myocardium. Despite this, the addition of cardioplegia caused the final functional recovery to be almost doubled and postischemic enzyme release to be almost halved. In considering the mechanisms by which hypothermia and cardioplegia confer protection upon the ischemic myocardium, it is accepted that hypothermia acts primarily by reducing metabolic rate and hence cellular energy requirements under the condition of restricted energy availability imposed by ischemia. In addition hypothermia slows various degradative processes, which further contributes to preventing the onset of irreversible damage. The protective effects of chemical cardioplegia are likely to be twofold: First, the inclu-

sion of agents like potassium to induce immediate diastolic arrest and prevent ischemic contraction conserves cellular energy supplies for tissue protection and functional recovery. Second, the inclusion of agents such as magnesium and potassium serves to combat one or more of the deleterious effects of tissue ischemia (such as cellular potassium and magnesium loss and cellular calcium uptake), thereby preventing or delaying the onset of irreversible cell damage. Thus from a theoretical standpoint it is perhaps not surprising that hypothermia and cardioplegia possess separate and additive protective properties. The second point to emerge from this study relates to the similarity in results between this study and the study in the preceding paper.! Despite major differences in the experimental model and the species under study, the results were qualitatively and quantitatively very similar and the conclusions were identical. This serves to reinforce our belief that the isolated perfused working rat heart preparation can act as an efficient and

Volume 79 Number 1

Additive effects of cardioplegia and hypothermia

43

January, 1980

valuable primary screen for the assessment of procedures designed to protect the myocardium during ischemic cardiac arrest. The assistance of Mrs. C. Boles is gratefully acknowledged as is the advice and discussion of Dr. F. Rosenfeldt. REFERENCES Rosenfeldt FL, Hearse Dl, Cankovic-Darracott S, Braimbridge MY: The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the dog. 1 THORAC CARDIOVASC SURG 79: 29-38, 1980 2 Bernhard WF, Schwartz HF, Mallick NP: Elective hypothermic cardiac arrest in normothermic animals. Ann Surg 153:43, 1961 3 Gott YL, Bartlett M, Johnson lA, Long DM, Lillehei CW: High energy phosphate levels in the human heart during potassium citrate arrest and selective hypothermic arrest. Surg Forum 10:54, 1960 4 Griepp RB, Stinson EB, Shumway NE: Profound local hypothermia for myocardial protection during open-heart surgery. 1 THORAC CARDIOVASC SURG 66:731, 1973 5 Proctor E: Early sinus rhythm in dog hearts preserved for 96 hours and assessed ex vivo. Transplantation 13:437, 1972 6 Engedal H, Skegseth E, Saetersdal S, Myklebust E: Cardiac hypothermia evaluated by ultrastructural studies in man. 1 THORAC CARDIOVASC SURG 75:548, 1978 7 Gay WA, Ebert PA: Functional metabolic and morphologic effects of potassium induced cardioplegia. Surgery 74:284, 1973 8 Bretschneider Hl, Hubner G, Knoll D, Lohr B, Nordbeck H, Spieckermann PG: Myocardial resistance and tolerance to ischemia. Physiological and biochemical basis. 1 Cardiovasc Surg 16:241, 1975 9 Kirsch U, Rodewald G, Kalmar P: Induced ischemic arrest. Clinical experience with cardioplegia in open-heart surgery. 1 THORAC CARDIOVASC SURG 63:121,1972 10 Hearse DJ, Stewart DA, Braimbridge MY: Hypothermic arrest and potassium arrest. Metabolic and myocardial protection during elective cardiac arrest. Circ Res 36:481 , 1975 II Schaff HY, Dombroff R, Flaherty IT, Bulkley BH, Hutchins GM, Goldman RA, Gott YL: Effect of potassium cardioplegia on myocardial ischemia and postarrest ventricular function. Circulation 58:240, 1978 12 Behrendt DM, Jochim KE: Effect of temperature of cardioplegic solution. 1 THORAC CARDIOVASC SURG 76:353, 1978 13 Engelman RM, Levitsky S, O'Donoghue Ml, Auvil 1: Cardioplegia and myocardial preservation during cardiopulmonary bypass. Circulation 58Suppl I: 107, 1978 14 Harlan Bl, Ross E, MacManus Q, Knight R, Luber 1,

Starr A: Cardioplegic solutions for myocardial preservation. Analysis of hypothermic arrest, potassium arrest and procaine arrest. Circulation 58: Suppl I: 114, 1978 15 Weisel RD, Lipton tH, Lyall RN, Baird Rl: Cardiac metabolism and performance following cold potassium cardioplegia. Circulation 58:Suppl 1:217, 1978 16 Hearse DJ, 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, 1976 17 Hearse Dl, Stewart DA, Braimbridge MY: Myocardial protection during bypass and arrest: A possible hazard with lactate containing infusates. 1 THORAC CARDIOVASC SURG 72:880, 1976 18 lynge P, Hearse DJ, Braimbridge MY: Myocardial protection during ischemic cardiac arrest. A possible hazard with calcium-free cardioplegic infusates. 1 THORAC CARDIOVASC SURG 74:848, 1977 19 lynge P, Hearse Dl, deLeiris 1, Feuvray D, Braimbridge MY: Protection of the ischemic myocardium. Ultrastructural, enzymatic and functional assessment of the efficacy of various cardioplegic infusates. 1 THORAC CARDIOVASC SURG 76:2, 1978 20 Hearse DJ, Stewart DA, Braimbridge MY: Myocardial protection during ischemic cardiac arrest. Possible deleterious effects of glucose and mannitol in coronary infusates. 1 THORAC CARDIOVASC SURG 76:16, 1978 21 Hearse DJ, Stewart DA, Braimbridge MY: Myocardial protection during ischemic cardiac arrest. The importance of magnesium in cardioplegic infusates. 1 THORAC CARDIOVASC SURG 75:877, 1978 22 Langendorff 0: Untersuchungen am uberlebenden Saugetierherzen. Pfluegers Arch 61:291, 1895 23 Krebs HA, Henseleit K: Untersuchunger uber die HarnstoffbildUng im Tierkorper. Hoppe Seylers Z Physiol Chern 210:33, 1932 24 Umbreit WW, Burris RH, Stauffer IF: Preparation of Krebs-Ringer phosphate and bicarbonate solution, Manometric Techniques, Minneapolis, 1969, Burgess Publishing Compnay, p. 132 25 Wollenberger A, Ristau 0, Schoffa G: Eine einfache Technik der extremschnellen Abkuhlung grosser Gewbstucke. Pfluegers Arch 270:399, 1960 26 Hearse Dl, Stewart DA, Chain EB: Recovery from cardiac bypass and elective cardiac arrest. The metabolic consequences of various cardioplegic procedures in the isolated rat heart. Circ Res 35:448, 1974 27 Hearse Dl, Chain EB: The role of glucose in the survival and recovery of the anoxic isolated perfused rat heart. Biochem 1128:1125, 1972 28 Hearse Dl, Humphrey SM: Enzyme release during myocardial anoxia: A study of metabolic protection. 1 Mol Cell Cardiol 7:463, 1973