Optimal myocardial preservation with an acalcemic crystalloid cardioplegic solution

J

THORAC CARDIOVASC SURG

1987;93:838-46

Optimal myocardial preservation with an acalcemic crystalloid cardioplegic solution The effect of the calcium and oxygen contents of a hyperkalemic glucose--containing cardioplegic solution on myocardial preservation was examined in the isolated working rat heart. The cardioplegic solution was delivered at 4° C every 15 minutes during 2 hours of arrest, maintaining a myocardial temperature of 8° ± 2° C. Hearts were reperfused in the Langendorff mode for 15 minutes and then resumed the working mode for a further 30 minutes. Groups of hearts were given the oxygenated cardioplegic solution containing an ionized calcium concentration of 0, 0.25, 0.75, or 1.25 mmoljL or the same solution nitrogenated to reduce the oxygen content and containing 0 or 0.75 mmol ionized calcium per liter. The myocardial adenosine triphosphate concentrations at the end of arrest in these six groups of hearts were 15.6 ± 1.2, 9.5 ± 0.5, 8.2 ± 1.1, 4.9 ± 1.8, 10.1 ± 2.0, and 1.6 ± 0.4 nmol/mg dry weight, respectively. At 5 minutes of working reperfusion, the percentages of prearrest aortic flow were 80 ± 2,62 ± 4, 33 ± 6, 37 ± 5, 48 ± 7 and 46 ± 8, respectively. The differences among the groups in adenosine triphosphate concentrations and in functional recovery diminished during reperfusion. In hearts given the hypoxic calcium-containing solution, there was a marked increase in coronary vascular resistance during the administration of successive doses of cardioplegic solution, which was rapidly reversible upon reperfusion. These data indicate that hearts given the acalcemic oxygenated solution had better adenosine triphosphate preservation during arrest and better functional recovery than hearts in any other group. Addition of calcium to the oxygenated cardioplegic solution decreased adenosine triphosphate preservation and functional recovery. Oxygenation of the acalcemic solution increased adenosine triphosphate preservation and functional recovery. The lowest adenosine triphosphate levelsat end arrest were observed in hearts given the hypoxic calcium-containing solution. In the setting of hypothermia and multidose administration, the addition of calcium to a cardioplegic solution resulted in increased energy depletion during arrest and depressed recovery.

Brian R. Boggs, M.D.,* David F. Torchiana, M.D., Gillian A. Geffin, M.B., B.S., James S. Titus, Brian E. Redonnett, Dennis D. O'Keefe, M.D., John B. Newell, B.A., and Willard M. Daggett, M.D., Boston. Mass.

Long before the advent of cardioplegia or the inception of modern cardiac surgery, Ringer' noted that the isolated frog heart stopped beating in the absence of

From the Department of Surgery and the Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Mass. Supported in part by National Institutes of Health Grant HL 12777. Received for publication March 14, 1986. Accepted for publication June 25, 1986. Address for reprints: Willard M. Daggett, M.D., Department of Surgery, Massachusetts General Hospital, Boston, Mass. 02114. *Present address: Department of Surgery, University of Oklahoma Health Sciences Center, P.O. Box 23607, Oklahoma City, Okla. 73126.

838

calcium. In synergism with an elevated potassium concentration, calcium-free cardioplegic solutions produce rapid and prolonged cardiac arrest. 2 The safety of acalecemic cardioplegia has been open to question, however, and calcium is included in the widely used St. Thomas' Hospital solution) as well as in many other formulations. Calcium is added to avoid the calcium paradox'<-the massive cellular damage that occurs after even a brief period of normothermic acalcemic perfusion when calcium is readmitted. Supporting the prudence of this measure are studies demonstrating inferior myocardial preservation when acalcemic solutions are used experimentally.t' Hypothermia" and noncoronary collateral flow? protect against the calcium paradox when a calcium-free cardioplegic solution is used in the clinical setting. The

Volume 93 Number 6

Acalcemic cardioplegia

June 1987

Table I. Composition of solutions*

Na+ (mEq/L) K+ (mEq/L) Ca '" (mEq/L) Mg'" (mEq/L) CI- (mEq/L) HCO,- (mEq/L) H,PO,- (mEq/L) 50,-- (mEq/L) Glucose (rnmol/L) Mannitol (rnrnol/L)

Krebs-Henseleit bicarbonate buffer

Cardioplegic solution

143.0 5.9 2.4 2.4 125.1 25.0 1.2 2.4 11.0

109.3 20.0

102.4 26.8

839

Table II. Experimental groups: Cardioplegic solution modifications* Group

Gasi

Ionized calcium (mmol/L)

I 2 3 4 5

Oxygen Oxygen Oxygen Oxygen Nitrogen Nitrogen

0 0.25 0.75 1.25 0 0.75

6

*Furthcr details concerning the cardioplcgic solutions are in the text.

27.8 54.9

*In appropriate experimental groups. CaCl, was added to the cardioplegic solution (Table II)

laboratory studies cited above 3. 5 exclude these protective elements by using an isolated heart preparation and normothermic ischemia. Whereas noncoronary collateral flow is an uncertain and uncontrolled variable in cardiac operations, hypothermia is a most important and universally applied facet of cardioplegic myocardial protection. As pointed out by Yamamoto, Braimbridge, and Hearse,' an optimal concentration of calcium at 37° C may not be optimal at a lower temperature. Similarly, an optimal calcium concentration in one solution may not be best for another cardioplegic solution of otherwise different composition. Oxygenation" and multidose delivery? can further improve myocardial preservation with crystalloid cardioplegic solutions and represent additional variables that might affect the response to calcium. In this study we sought to examine the effect of varying the calcium and oxygen contents during hypothermic multidose administration of cardioplegic solution in the isolated rat heart. Metbods Hearts were obtained from male Sprague Dawley rats weighing 250 to 400 gm. Before cardiectomy the animals were heparinized (1,000 units intraperitoneally) and then anesthetized with pentobarbital (240 tug] kg). The isolated heart was mounted on the apparatus developed by Yamamoto, Braimbridge, and Hearse' and initially perfused for 15 minutes via the aortic root at a pressure of 100 cm H 20 by the method of Langendorff with Krebs-Henseleit bicarbonate buffer (Table I) at st: C, gassed with 95% oxygen and 5% carbon dioxide. All solutions in this study were passed through a 5 ~m porosity filter before administration and bubbled with the appropriate gas mixture containing carbon dioxide before addition of calcium to avoid

tThe equilibrating mixture consisted of 98(it. of the specified gas and 2% carbon dioxide.

precipitation of calcium in bicarbonate-containing solutions. During the Langendorff perfusion period the left atrium was cannulated. A standard working heart preparation to was then employed by perfusing the left atrium at a pressure of 15 ern H 20 and allowing the left ventricle to eject against a pressure of 100 em H 20 into a recirculating aortic column. Aortic flow was measured by a flowmeter in the aortic column. Coronary flow was measured by timed volumetric collection of effluent from the right side of the heart. Heart rate was obtained from a strip-chart recording of aortic pressure. Aortic flow, coronary flow, and heart rate were determined at 5 minute intervals during the 20 minute working control period. Prearrest control values for these variables were obtained by averaging the data collected at 10, 15, and 20 minutes. Hearts with aortic flows less than 40 ml/rnin, apparent coronary flows greater than 24 ml/ min, or a heart rate of less than 200 beats/min were rejected from the study. In additional control studies with hearts in the working mode, aortic flow was maintained for 2 hours at greater than 90% of its initial stable value. At the end of the control period the heart was rendered globally ischemic by clamping the aortic and atrial catheters. Arrest was immediately induced with a hyperkalemic, crystalloid cardioplegic solution, which was delivered at a constant pressure of 65 em H 20 at 4 0 C via a side arm on the aortic cannula. Cardioplegic solution was intermittently infused in this manner every 15 minutes for 2 hours. The volume of coronary effluent was 15 ml for the first administration of the solution and 10 ml for each subsequent administration. The time required for each cardioplegic infusion was recorded. During the arrest period the heart was maintained at 8 ° ± 2 ° C by means of a water-jacketed chamber surrounding the heart. The heart was reperfused with Krebs-Henseleit buffer at 37° C in the Langendorff mode for 15 minutes and then returned to the working mode for 30 minutes, during which time the recovery of

The Journal of Thoracic and Cardiovascular

8 4 0 Boggs et al.

Surgery

Table Ill, Hemodynamic variables before arrest and as percentage recovery during reperfusion Reperfusioni (% of prearrest value) Group

n 12

Variable

AF CF

HR

2

8

AF CF

HR

3

12

4

8

AF CF

HR

AF CF

HR

5

8

AF CF

HR

6

8

AF CF

HR

Prearrest* 58 18 283 55 18 299 61 17 273 61 18 312 67 19 298 64 17 291

± 3 ml/rnin ± I ml/rnin ± 8 beats/min

± 2 ml/rnin ± I nil/min ± 19 beats/min

± 3 ml/rnin ± I ml/rnin ± 12 beats/min ± 2 ml/rnin ± I ml/rnin ± 13 beats/min ± 4 ml/rnin

± I ml/rnin ± 9 beats/min

± 3 ml/rnin ± I mljmin ± 8 beats/min

5 min 80 90 108 62 98 107 33 91 100 37 90 100 48 90 102 46 90 104

± 2 ± 3 ± 2 ± 4:1:

± 6 ± 3

± 6§ ± 5 ± 2 ± 5§

± 5 ± 2 ± 7§ ± 4

± 4 ± 8 ± 4 ± 4

I

10 min 84 94 107 71 100 105 43 87 105 45 89 94 53 93 102 47 92 108

± ± ± ± ± ±

2 3 2

5

5 3 ± 5§ ± 5 ± 3 ± 5§ ± 8 ± 4 ± 7§ ± 3 ± 6

± 8

± 5

± 5

I

15 min 87 ± 2 94 ±. 2 106 ± 2 84 ± 3 98 ± 5 105 ± 2 55 ± 4§ 87 ± 4 102 ± 3 55 ± 6§ 92 ± 7 100 ± 3 62 ± 7§ 91 ± 3 101 ± 5 52 ± 8 96 ± 5 105±4

1

20 min 88 96 104 78 97 103 59 90 103 61 89 99 67 94 103 55 101 105

± 2 ± 3 ± 2

± 4 ± 5 ± 3 ± 4§ ± 3 ± 3 ± 6§

± 6 ± 2

± 511 ± 4 ± 5 ± 6 ± 6 ± 4

1

25 min 88 ± 97 ± 105 ± 77 ± 100 ± 106 ± 64 ± 88 ± 101 ± 64 ± 94 ± 100 ± 67 ± 98 ± 101 ± 55 ± 102 ± 104 ±

2 3

2 5:1: 4 3 5§ 4 4 3§

6 2 ~I

4 4 5 6 5

1

30 min

87 99 104 85 102 104 62 90 103 65 96 99 68 98 101 57 105 103

± 2 ± 3 ± 2 ± 3 ± 5 ± 3 ± 5§ ± 3 ± 3 ± 5§ ± 5 ± 3 ± ± ± ±

~I

5 4

5 ± 7 ± 5

Legend: Values are means ± standard error of the mean. AF, Aortic flow. CF, Coronary flow. HR, Heart rate.

'The prearrest values are the average of data obtained after 10, 15, and 20 minutes in the working mode. tThe working mode was preceded by 15 minutes in the Langendorff mode. :j:p

< 0.05 versus Group 1.

lIP < 0.005

versus Group 1. §p < 0.0005 versus Group I.

aortic flow, coronary flow, and heart rate was assessed at 5 minute intervals. Before the cardioplegic solution was infused, at the end of the arrest period, and at the end of reperfusion, parallel groups of hearts were subjected to assay of myocardial high-energy phosphate concentrations and heart water. Approximately 100 mg of tissue was excised from the apex, rapidly frozen by compression between metal paddles at -70° C, and then divided into three portions. Each portion was assayed for adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and creatine phosphate (CP) by high-pressure liquid chromatography as previously described. I I The averaged results were expressed as nanomoles per milligram of dry weight using the appropriate mean heart water of each experimental group. Total adenine nucleotides (TAN) were calculated as the sum of ATP, ADP, and AMP. Heart water, determined by desiccation at 90° C to a constant weight, was obtained in two samples taken from the remainder of the ventricles and the results were averaged. Cardioplegic solutions. The composition of the basic cardioplegic solution is shown in Table I. Varying amounts of calcium chloride were added to the base

cardioplegic solution and the ionized calcium concentration of each solution was determined (Nova 2 calcium ion selective electrode, Nova Biochemical, Newton, Mass.). Before delivery to the heart, the cold cardioplegic solution was gassed with either 98% oxygen and 2% carbon dioxide or 98% nitrogen and 2% carbon dioxide. This gave an arterial oxygen tension above 500 mm Hg or below 50 mm Hg, respectively, when measured at 10° C, and a pH of 7.33 to 7.53 measured at 37° C. The equilibrating gas and ionized calcium concentration in the cardioplegic solution in each of the six experimental groups are shown in Table II. The sequence of experiments was determined by mixing pairs of hearts from each group into a randomized block design. All rats received humane care in compliance with the "Guiding Principles in the Care and Use of Animals" approved by the Council of the American Physiological Society (revised 1980) and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication No. 78-83, revised 1978). Data were entered into a computer (Vax 11/780, Digital Equipment Corporation, Maynard, Mass.) for storage and analysis. The data were subjected to twoway analysis of variance (ANOVA) by the BMDP

Volume 93

Acalcemic cardioplegia 8 4 1

Number 6 June 1987

100,----------------------------,

~

~ ~

80

~

70

i=:::

'<:{

......

t3

60

'<:{

50

~

40

~

~

Group

90

°

__..&---O'----6---....}, 1 02. No Co"

2

2,

tt

0.25mM Co

2

4 3

30

5

10

Fig. 1. Recovery of aortic flow during reperfusion, expressed as a percentage of prearrest aortic flow. Rat hearts were subjected to 2 hoursof cardiop1egic arrest and 15 minutes of reperfusion in the Langendorff mode. This was followed by conversion to the working mode. The abscissa indicates duration of reperfusion in the working mode. The numberof hearts represented by each data point ranges from 8 to 12. Bars indicate ± standard error of the mean. Statistical Software program BMDP4V.12 Mean coronary vascular resistance measured during each cardioplegic dose and each parameter of left ventricular performance, expressed as a percentage of its prearrest value, were treated separately in a univariate repeated measures analysis. Percent heart water and myocardial concentrations of adenine nucleotides and CP were subjected to two-way ANOVA with experimental group and time (end arrest and end reperfusion) as betweensubjects factors. For comparison of prearrest control values of these variables with data obtained at end-arrest and end reperfusion, this analysis was performed with the prearrest data entered in each of the experimental groups. When there were strong interactions between the main effects, data at single levels of the trial factor or between-subjects factors were subjected to one-way ANOVAs. When the ANOV As rejected the hypothesis of equal means, the following null hypotheses were tested by examining the significance of the differences between specific pairs of means: that the addition of calcium at any concentration had no effect in oxygenated cardioplegic solution (Group 1 versus each of Groups 2, 3, and 4) or in hypoxic cardioplegic solution (Group 5 versus Group 6); that there were no differences between oxygenated and hypoxic cardioplegic solutions whether the solutions were acalcemic or calcium containing (Group 1 versus Group 5, Group 3 versus Group 6). Differences were considered significant at

p < 0.05. All data are expressed as mean ± standard error of the mean.

Results Ventricular function. The values for aortic flow, coronary flow, and heart rate obtained before and after arrest are shown in Table III. Fig. 1 displays recovery of aortic flow, expressed as a percentage of prearrest aortic flow, for each of the six groups. Group 1 hearts, which received the oxygenated acalcemic cardioplegic solution, recovered 87% of control aortic flow at 30 minutes and had better preservation of ventricular function when compared with all other groups. The percentage of prearrest aortic flow was significantly greater in Group 1 than in Group 2 at 5 and 25 minutes of reperfusion and very significantly greater throughout reperfusion than in either Group 3 or 4. These results indicate that addition of calcium to the oxygenated cardioplegic solution reduced the level of recovery, with the higher concentrations having a greater effect. Oxygenation of the acalcemic cardioplegic solution markedly improved recovery. This improvement was highly significant, but improvement by oxygenation in the presence of calcium did not reach statistical significance. There was a significant overall improvement in aortic flow during reperfusion. There were no differences among the groups in the percentage recovery of coronary flow, although there

The Journal of

8 4 2 Boggs et al.

Thoracic and Cardiovascular Surgery

Table IV. Myocardial concentrations of adenine nucleotides and creatine phosphate (nmolfmg dry weight)* 2 hr Arrest Group

2 3 4 5 6

ATP

ADP

AMP

TAN

CP

15.6 ± 1.2 (7) 9.5 ± O.Wll (4) 8.2 ± 1.1*'ll** (6) 4.9 ± 1.8*'ll (4) \O.l ± 2.0*'ll** (5) 1.6 ± 0.4* (4)

8.1 ± 0.2* (7) 8.4 ± 0.7 (4) 8.3 ± 0.7§** (6) 7.3 ± (4) 8.0 ± 0.5§** (5) 2.9 ± 0.1* (4)

3.7 ± 0.5§ (7) 6.2 ± 0.8 (4) 9.9 ± 1.2*'ll** (6) 12.9 ± 2.n'll (4) 6.6 ± 1.6** (5) 17.6 ± 0.8* (4)

27.4 ± I.I§ (7) 24.1 ± 1.8* (4) 26.5 ± 1.4§ (6) 25.1 ± 0.9* (4) 24.8 ± 0.8* (5) 22.0 ± 0.5* (4)

22.4 ± 1.6* (5) 11.2 ± 0.9'll (4) 9.3 ± l.7'lltt (6) 9.8 ± 1.5'll (4) 6.9 ± 0.5 (3) 4.9 ± 0.7* (4)

O.n

Legend: Values are means ± standard error of the mean. The number of hearts represented by each mean is given in parentheses below it. ATP. Adenosine triphosphate. ADP, Adenosine diphosphate. AMP, Adenosine monophosphate. TAN, Total adenine nucleotides. CP, Creatine phosphate. "Prearrcst values were ATP = 15.3 ± 0.8, ADP = lOA ± 0.7, AMP = 5.9 ± 0.6, TAN = 31.6 ± 0.6, TAN tFifteen minutes of reperfusion in the Langendorff mode followed by 30 minutes in the working mode. #p

< 0.05 versus Group

= 31.6 ±

1.6 (n

= 10), CP = 11.3 ±

1.2 (n

= 7).

I.

< 0.006

versus Group I. ttp < 0.05 versus Group 6. "p < 0.006 versus Group 6. §p < 0.05 versus prearrest. +p < 0.0 I versus prearrest. ~p

lIP < 0.005

versus 2 hr arrest.

Table V. Heart water* 2 hr Arrest

45 min Reper!usiont

Heart water Group

n

I 2 3 4 5 6

5 4 5 4 4 4

I

(%) 83.7 81.1 81.5 81.1 82.7 78.9

± ± ± ± ± ±

0.3 0.3*§ 0.5*'ll# O.I*§ 0.5'll O.l§#

n II 8

10 7 6 5

I

Heart water (%) 84.3 84.2 83.8 84.1 84.6 84.6

± ± ± ± ± ±

0.3 0.211 0.3** 0.211 0.2** 0.111

Legend: Values are means ± standard error of the mean. 'Prearrest heart water was 83.0% ± 0.2% (n = 8). tFifteen minutes of reperfusion in the Langendorff mode followed by 30 minutes in the working mode. +p < 0.0005 versus Group I. ~p < 0.0002 versus Group 6. #p < 0.02 versus prearrest.

§p < 0.0002 versus prearrest. **p < 0.001 versus 2 hr. arrest.

lIP < 0.000 I versus

2 hr. arrest.

was a significant overall increase in coronary flow during reperfusion. There were no significant differences among the groups or over time in the percentage recovery of heart rate. High-energy phosphates. Table IV shows the myocardial concentrations of adenine nucleotides and CP

before cardioplegic arrest and for all groups at the end of the arrest period and at the end of reperfusion. After 2 hours of arrest, only the acalcemic oxygenated cardiaplegic solution maintained myocardial ATP at the prearrest level (15.3 ± 0.8 nmoljmg dry weight). With both oxygenated and hypoxic cardioplegic solutions, when calcium was added the end-arrest tissue ATP levels decreased significantly compared to ATP in the group of hearts given the corresponding acalcemic cardioplegic solution. Tissue AMP levels, in contrast to ATP, increased as calcium was added; these increases in AMP were all significant except the effect of adding calcium in a concentration of 0.25 mmol/L to the oxygenated cardioplegic solution, With both acalcemic and calcium-containing solutions, the oxygenated cardioplegic solution preserved ATP and ADP to a greater degree than the respective hypoxic solutions; these differences were highly significant. With the hypoxic calcium-containing solution the ATP concentration was only 1.6 ± 0.4 nmoljmg dry weight at end arrest. There were no differences in end-arrest TAN among the groups, although TAN was significantly less than its prearrest value in all groups. By the end of reperfusion, the differences in adenine nucleotides diminished: There were no differences among the groups in ATP, ADP, or AMP. TAN was unchanged from end arrest.

Volume 93 Number 6

Acalcemic cardioplegia 8 4 3

June 1987

45 min reperfusionf ATP

ADP

10.1 ± O.~I (11) 10.7 ± 0.6 (8) 10.8 ± 1.0 (12) 8.0 ± 0.5 (9) 9.3 ± 1.0 (8)

9.0 ± 0.2 (II) 8.5 ± 0.4 (8) 8.4 ± 0.5 (12) 7.9 ± 0.3 (8) 8.6 ± 0.6 (8) 7.9 ± 0.511 (8)

7.8 ± 1.11! (8)

AMP

6.7 ± 0.11 (II) 5.9 ± 0.8 (8) 6.1 ± 0.11 (12) 6.8 ± 0.811 (8) 5.8 ± 0.7 (8) 7.9 ± 1.~1 (8)

After 2 hours of arrest, the CP concentration was significantly greater than before arrest in those hearts that received oxygenated acalcemic cardioplegic solution and significantly depressed in those hearts that received hypoxic calcium-containing cardioplegic solution. The addition of calcium to oxygenated cardioplegic solutiondecreased end-arrest CPo After reperfusion, CP decreased greatly in the hearts given oxygenated acalcemic solution and increased greatly, as compared to the end-arrest values, in the hearts given hypoxic calciumcontaining cardioplegic solution. Heart water. Table V shows the percentage heart water before arrest, at the end of arrest, and at the end of reperfusion. After 2 hours of arrest, hearts in Group 6, given the hypoxic calcium-containing cardioplegic solution, had significantly less heart water than either hearts given oxygenated cardioplegic solution with the same calcium concentration or hearts given the hypoxic acalcemic solution. Heart water increased significantly in Groups 2 to 6 during reperfusion. There was no difference between Group 6 and the other groups after reperfusion. Coronary vascular resistance. Fig. 2 shows the number of seconds required to deliver a single milliliter of cardioplegic solution during each of the eight cardioplegic administrations (total time to deliver dose divided by total volume). Hearts in Group 6 had a pronounced increase in coronary vascular resistance during the arrest period; the final administration of hypoxic calcium-containing cardioplegic solution took three times as long as the final dose of the other solutions. Coronary vascular resistance in Group 6 during the final dose was significantly greater than in Group 5, the group that most closely approached it (p < 0.0001). Coronary vascular resistance in Group 6 was also significantly

TAN

CP

25.8 ± 0.9 (II) 25.1 ± 1.0 (8) 25.2 ± 1.1 (12) 22.7 ± 0.6 (8) 23.7 ± 1.1 (8) 23.7 ± 1.4 (8)

8.2 ± I.~I (9) 13.3 ± 1.3# (7) 16.0 ± 2.1# (II) 14.2 ± 1.9# (5) 9.2 ± 3.6tt (3) 17.6 ± 0.711 (3)

greater during the final than during the initial dose (p < 0.0001). Discussion Earlier canine studies from this laboratory have utilized prolonged periods of ischemia and intermittent administration to establish the efficacy of oxygenating cardioplegic solutions." Results in similar canine studies support the inclusion of calcium 13 in the same cardioplegic solution as used in this report. Our present results with the isolated rat heart confirm the salutory effect of oxygenation but do not favor the inclusion of calcium. In this study oxygenated acalcemic cardioplegic solution preserved a higher level of left ventricular function and myocardial ATP concentration than hypoxic acalcemic solution. This accords with the beneficial effect of oxygenation on the preservation of function- 14, 15 and ATP concentratiorr-" described previously by various authors. In contrast to our previous work in the dog, addition of calicum had deleterious effects.13 Five minutes after resuming the working mode upon reperfusion, the hearts that received oxygenated acalcemic cardioplegic solution recovered 80% of prearrest aortic flow. Incremental addition of calcium to the oxygenated basic cardioplegic solution reduced recovery of left ventricular function, as shown in Fig. 3. A similar relationship between calcium content of cardioplegic solution and ATP levels at the end of the ischemic period appears in Fig. 4. In the hypoxic groups, calcium also depressed ATP concentration at this time. Postischemic ATP levels and functional recovery can be dissociated experimentally. In experiments in the isolated rat heart, Neely and Grotyohann" have shown that lactate accumulation during an ischemic period can profoundly depress functional recovery. When hearts

The Journal of Thoracic and Cardiovascular Surgery

8 44 Boggs et al.

---.. (rj


<,

Group 24 22

~

~

20

~

18

~ ~

16

~

tj

Q;:

Cl::

1

0--0

2

o--a

3

6----D.

4 5 6

"<1--'il

.........

....-

°°

2 I No Co++

2 • 0.25 mM

02. 02. N2 , N2

12

§

!O

::.... Cl::

~ ~

8

//1

I

I

I

/f/

l

I

/

/

I I

I

14

::5

1

0.75mM Co·· 1.25mM Co" No Co·· 0.75 mM Co··

"'l:

~

++

Co

/

.J-/1

l..__ -s- _--1

8 6

~1

°

I

2

I

3

I

4

I

5

6

7

8

CARDIOPLEGIA DOSE NUMBER

Fig. 2. Mean coronary vascular resistanceduring cardioplegic solution (eS) administration. Cardioplegic solutions were administered every 15 minutes for 2 hours. The first (arresting) dose of cardioplegic solution was 15 ml and the subsequent doses were each 10 ml. The number of hearts represented by each data point ranges from 12 to 19. Bars indicate ± standard error of the mean. For clarity, only selected error bars are shown.

depleted of glycogen before arrest were compared to normal hearts, a similar duration of ischemia resulted in a lower level of lactate buildup and better functional recovery despite far lower stores of ATP. No doubt there are other detrimental aspects of ischemia that can be isolated under particular experimental circumstances. In the present study functional recovery correlated well with end-arrest ATP levels, a relationship others have shown previously. I? In our experimental model calcium reduces ATP stores during ischemia in a dose-dependent manner. The presence of calcium in the cardioplegic solution, therefore, alters the balance between ATP production and ATP consumption during the arrest period. Both an increased ATP consumption and an inhibition of ATP production are theoretically possible because increased intracellular calcium can stimulate the ATPase activities associated with ion pumps" and myofibrils" and can inhibit mitochondrial oxidative phosphorylation." It has

been suggested that the energy utilization of the arrested heart is primarily determined by the level of activity of the contractile system, with a lesser contribution to energy consumption by membrane ion pumps and other energy-consuming cellular functions." The conservation of ATP we observed with acalcemic cardioplegic solution may reflect maximal deactivation of the contractile elements. As increasing amounts of calcium are supplied to the ischemic myocardium, there may be an increase in the resting activity of the muscle despite maintained electrical arrest. Consumption of high-energy phosphate stores with diminished ventricular functional recovery could then follow. As Iowa calcium concentration as 0.05 mmol/L can eliminate the calcium paradox." Hence a very small amount of inadvertently included calcium can alter the behavior of what is intended to be an acalcemic solution. Potential sources of protective calcium for the myocardium are noncoronary collateral flow? and calcium contamination of the cardioplegic solution. Noncoronary flow is not a consideration in the isolated heart model we used. To confirm that calcium contamination was not present in our cardioplegic solution, we perfused four hearts with the acalcemic cardioplegic solution at 37° C for 10 minutes. Calcium-containing KrebsHenseleit buffer was then reintroduced for 20 minutes and the coronary effluent collected. The hearts became pale and failed to demonstrate any sign of mechanical recovery. Creatine kinase release from the hearts averaged 935 units/gm of dry myocardial tissue. These findings are consistent with published descriptions of the calcium paradox" and confirm that our acalcemic cardioplegic solution was calcium free. Previous reports have shown protection against calcium paradox injury by temperatures less than 28 ° C6 or calcium-free infusions of short duration.' Hypothermia and intermittent infusion of cardioplegic solution, as utilized both in this experiment and clinically, minimize the likelihood of the calcium paradox. Hearts that received a hypoxic calcium-containing cardioplegic solution (Group 6) had a progressive increase in coronary vascular resistance toward the end of the arrest period (Fig. 2). This increase in coronary vascular resistance seems paradoxic, because hypoxemia is a potent coronary vasodilator." As indicated by the percentage of heart water at the end of ischemia, hearts in Group 6 showed no greater tendency toward tissue edema, which might cause extrinsic compression of the coronary vessels, than hearts in the other groups (Table V). The increased resistance in Group 6 was fully reversible on reperfusion as coronary flow returned to

Volume 93 Number 6

Acalcemic cardioplegia

June 1987

845

100

~ It

80

~

h::

~

60

"'l:

~

)...

~ :s:

40

~

20

8 ~

0

Groupna [Co+fj(mMJ

1 0

2 0.25

3 0.75

4 1.25

OXYGEN

5 0

6 0.75

NITROGEN

Fig. 3. Functional recovery 5 minutes after resuming the working mode. Recovery is expressed as percent of prearrest aortic flow. Bars indicate ± standard error of the mean.

16

....

......... -t:: .~ III ~

~

"l:l

I::lI

~

\I')

~

14 12 10 8

<::>

~

6

~

4

~ "'l:

2 0

Group no. [eo H) (mM)

1 0

2 025

3 075

OXYGEN

4 1.25

5 0

6 075

NITROGEN

Fig. 4. Myocardial adenosine triphosphate (ATP) concentrations at the end of 2 hours of cardioplegic arrest. Bars indicate ± standard error of the mean.

the prearrest rate (Table III). The rapid reversibility of this phenomenon suggests that myocardial or vascular injury is not its cause. In conclusion, with multidose hypothermic cardia-

plegic solution in the isolated rat heart, the addition of calcium results in increased energy depletion during arrest and depressed recovery. Hypoxic calciumcontaining cardioplegic solution produces a reversible

The Journal of Thoracic and Cardiovascular

8 4 6 Boggs et al.

increase in coronary vascular resistance. The effect of calcium is possibly due to an increase in energy consumption associated with increased resting muscle tone. We wish to thank Alvin Denenberg for the high-pressure liquid chromatographic and other chemical analyses. We would also like to thank Douglas Malnati and James Vath, who assisted with the chemical analyses, Cyrus Noble and William Marasco, for technical assistance, and Victoria Groomes, Mary Chasse, and Judy Feiner, for preparing the manuscript. REFERENCES I. Ringer S. A further contribution regarding the influence of the differ~nt constituents of the blood on the contraction of the heart. J Physiol (Lond) 1882;4:29-42. 2. Hearse DJ, Braimbridge MV, Jynge P. Protection of the ischemic myocardium. In: Cardioplegia. New York: Raven Press, 1981:152-4. 3. Yamamoto F, Braimbridge MV, Hearse DJ. Calcium and cardioplegia: the optimal calcium content for the St. Thomas' Hospital cardioplegic solution. J THORAC CARDIOVASC SURG 1984;87:908-12. 4. Zimmerman ANE, Hiilsmann WC. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 1966;211:646-7. 5. Jynge P. Protection of the ischemic myocardium: calciumfree cardioplegic infusates and the additive effects of coronary infusion and ischemia in the induction of the calcium paradox. Thorac Cardiovasc Surg 1980;28:3039. 6. Holland CE, Olson RE. Prevention by hypothermia of paradoxical calcium necrosis in cardiac muscle. J Mol Cell Cardiol 1975;7:917-28. 7. Brazier J, Hottenrott C, Buckberg G. Noncoronary collateral myocardial blood flow. Ann Thorac Surg 1975;19:426-35. 8. Bodenhamer RM, DeBoer LWV, Geffin GA, et al. Enhanced myocardial protection during ischemic arrest: oxygenation of a crystalloid cardioplegic solution. J THORAC CARDIOVASC SURG 1983;85:769-80. 9. Engelman RM, Auvil J, O'Donoghue MJ, Levitsky S. The significance of multidose cardioplegia and hypothermia in myocardial preservation during ischemic arrest. J THORAC CARDIOVASC SURG 1978;75:555-63. 10. Neely JR, Rovetto MJ. Techniques for perfusing isolated

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