Enhanced myocardial protection with high-energy phosphates in St. Thomas’ Hospital cardioplegic solution

J

THORAC CARDIOVASC SURG

1987;93:415-27

Enhanced myocardial protection with high-energy phosphates in 81. Thomas' Hospital cardioplegic solution Synergism of adenosine triphosphate and creatine phosphate The potential for improving myocardial protection with the high-energy phosphates adenosine triphosphate and creatine phosphate was evaluated by adding them to the St. Thomas' Hospital cardioplegic solution in the isolated, working rat heart model of cardiopulmonary bypass and ischemic arrest. Dose-responsestudies with an adenosine triphosphate range of 0.05 to 10.0 mmol/L showed 0.1 mmol/L to be the optimal concentration for recovery of aortic flow and cardiac output after 40 minutes of normothermic (370 C) ischemic arrest (from 24.1 % ± 4.4% and 35.9% ± 4.1 % in the unmodified cardioplegia group to 62.6% ± 4.7% and 71.0% ± 3.0%, respectively, p < 0.001). Adenosine triphosphate at its optimal concentration (0.1 mmol/L) also reduced creatine kinase leakage by 39 % (p < 0.001~ Postischemic arrhythmias were also significantly reduced, which obviated the need for electrical defibrillation and reduced the time to return of regular rhythm from 7.9 ± 2.0 minutes in the control group to 3.5 ± 0.4 minutes in the adenosine triphosphate group. Under more clinically relevant conditions of hypothermic ischemia (200 C, 270 minutes) with multidose (every 30 minutes) cardioplegia, adenosine triphosphate addition improved postischemic recovery of aortic !low and cardiac output from control values of 26.8% ± 8.4% and 35.4% ± 6.3% to 58.0% ± 4.7% and 64.4% ± 3.7% (p < 0.01), respectively, and creatine kinase leakage was significantly reduced. Parallel hypothermic ischemia studies (270 minutes, 20 0 C) using the previously demonstrated optimal creatinine phosphate concentration (10.0 mmoljL) gave nearly identical improvements in recovery and enzyme leakage. The combination of the optimal concentrations of adenosine triphosphate and creatine phosphate resulted in even greater myocardial protection; aortic flow and cardiac output improved from their control values of 26.8 % ± 8.4% and 35.4% ± 6.3% to 79.7% ± 1.1 and 80.7% ± 1.0% (p < 0.001), respectively. In conclusion, both extraceUular adenosine triphosphate and creatine phosphate alone markedly improve the cardioprotective properties of the St. Thomas' Hospital cardioplegic solution during prolonged hypothermic ischemic arrest, but together they act additively to provide even greater protection.

Lary A. Robinson, M.D., Mark V. Braimbridge, F.R.C.S., and David J. Hearse, D.Sc.,

London, England

COld chemical cardioplegic solutions in cardiac surgery have greatly enhanced protection during ischemia From The Heart Research Unit, The Rayne Institute, St. Thomas' Hospital, London SEI, England. Supported in part by grants from the British Heart .Foundation and the St. Thomas' Hospital Research Endowments Fund. Received for publication Oct. IS, 1985. Accepted for publication Oct. 31, 1986. Address for reprints: Lary A. Robinson, M.D., Section of Thoracic and Cardiovascular Surgery, University of Nebraska Medical Center, 42nd and Dewey Ave., Omaha, Neb. 68105.

by slowing destructive myocardial changes, with a net improvement in patient survival.':' Many studies have demonstrated a strong correlation between preservation of the cellular content of high-energy phosphates (HEP), particularly adenosine triphosphate (ATP) and creatine phosphate (CP), and reversibility of the ischemic insult. '-3, 5-7 Over the past decade, numerous pharmacologic interventions have been developed to maintain or replete cellular high-energy stores by acting directly on degradative, salvage, or synthetic pathways of adenine nucleotide metabolism. Various substrates, precursors, and 415

The Journal of Thoracic and Cardiovascular

416 Robinson, Braimbridge, Hearse

Surgery

Table I. The St. Thomas' Hospital cardioplegic

solution (without procaine)* Compound Sodium chloride Potassium chloride Magnesium chloride Calcium chloride Sodium bicarbonate

Concentration (mmoljL) 110.0 16.0 16.0 1.2

10.0

pH adjusted to 7.8 Osmolarity = 324 mOsm H 20 "From Robinson, Braimbridge, and Hearse. Creatine phosphate: an additive myocardial protective and antiarrhythmic agent in cardioplegia. J THoRAc CARDIOVASC SURG 1984;87:190-200; by permission of The C. V. Mosby Company.

enzyme inhibitors of the purine metabolic pathways have been added to preischemic or postischemic coronary infusates, usually with favorable results." 8-22 ATP and CP themselves have also been administered with uniformly enhanced myocardial protection.P" Recent studies in this laboratory demonstrated that CP added at its optimal dosage to the St. Thomas' Hospital cardioplegic solution exerted potent protective and antiarrhythmic effects during prolonged hypothermic myocardial ischemia." Other reports of the successful use of HEP in shock and postischemic renal or hepatic injury31.32 suggest that the protective properties of CP may be mediated via ATP. If so, the inclusion in cardioplegic solutions of ATP, alone or in combination with CP, should also improve myocardial protection. The present study was designed with three aims: (1) to determine if ATP, exogenously administered with a cardioplegic solution, improved tolerance to myocardial ischemia; (2) to define ATP dose-response characteristics; and (3) to evaluate the combination of exogenous CP and ATP to enhance myocardial protection further. Materials and methods Experimental preparations. Hearts were obtained from male Sprauge-Dawley rats (200 to 250 gm body weight). In all studies, animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). The isolated, perfused, working rat heart preparation was used in this study, and this has already been described in detail elsewhere.v" Briefly, in this left

heart preparation, oxygenated perfusion medium (Krebs-Henseleit bicarbonate buffer,36,37 pH 7.4, containing glucose ILl mmol/L and gassed with 95% oxygen and 5% carbon dioxide) at 37° C enters the cannulated left atrium at a pressure of 17 em H 20. The perfusate passes into the left ventricle, from which it is spontaneously ejected via an aortic cannula against a hydrostatic pressure of 100 em H 20. Electrical pacing was not used in this study. Coronary effluent, flowing from the right atrium and ventricle, can be sampled for biochemical analysis or recirculated with the aortic outflow. Total cardiopulmonary bypass, maintaining coronary perfusion, is simulated by clamping the left atrial cannula and introducing perfusion fluid at 37° C into the aorta from a reservoir located 100 cm 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 by clamping the aortic cannula. During ischemia the heart may be maintained at 37° C, or any degree of hypothermia, by the use of heating or cooling circuits supplying the water-jacketed heart chamber. Short periods of preischemic coronary infusion (at any degree of hypothermia) of a cardioplegic solution may be achieved by use of a reservoir (located 60 em above the heart) attached to a side arm of the aortic cannula. Cardioplegic solutions. The composition of the basic St. Thomas' Hospital cardioplegic solution used in these studies is shown in Table 1,39 The solution was not oxygenated and did not contain procaine or lidocaine. The added concentration of ATP (Rona Laboratories Ltd, Hitchen, England) was varied in the range of 0 to 10 mmol/L. Small amounts of sodium hydroxide were added to the solution with increasing amounts of ATP so as to maintain the pH of the test solution at 7.8; no significant change in sodium concentration of the base solution occurred with these small sodium hydroxide additions. CP (Schiapparelli Pharmaceuticals, Torino, Italy) was added only in the hypothermia study (10 mmol/L), and the sodium concentration of the solution was adjusted to maintain the final sodium content at 120 mmoljL. Precautions were taken to prevent precipitation of calcium, and all solutions were filtered through a cellulose acetate membrane (pore size 5 [.Lm) just before use. Experimental time course (Fig. 1). Normothermia studies. Immediately after excision of the heart, the aorta was cannulated and Langendorff perfusion was initiated for a 5 minute washout period. Left atrial cannulation was accomplished during this 5

Volume 93

Myocardial protection with HEP

Number 3

March 1987

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Fig. 1. Experimental time course: Normothermia series. Hearts were perfused for 5 minutes in the Langendorff nonworking mode, after which they were converted to working preparations in which control functional indices were recorded for 20 minutes. After 3 minutes of cardioplegic infusion, hearts were subjected to 40 minutes of normothermic (37 C) ischemia. Hearts were then reperfused in the Langendorff mode for 15 minutes with collection of the coronary effluent for creatine kinase (CK) determination. Finally hearts were converted to the working mode, and postischemic functional recovery was monitored for 20 minutes. (From Robinson, Braimbridge, and Hearse. Creatine phosphate: an additive myocardial protective and antiarrhythmic agent in cardioplegia. J THORAC CARDIOVASC SURG 1984;87:190-200; by permission of the C. V. Mosby Company.) 0

minute period. The perfusion fluid during this period and subsequent perfusion periods was Krebs-Henseleit bicarbonate buffer (37° C). The heart was then converted to a working preparation by terminating retrograde aortic perfusion and initiating left atrial perfusion. During a 20 minute period, control values for aortic and coronary flow rates, peak aortic pressure, heart rate, and electronically differentiated aortic dP/ dt were recorded. At the end of the control period the atrial and aortic cannulas were clamped and the heart was subjected to a 3 minute period of normothermic (37° C) coronary infusion with the St. Thomas' Hospital cardioplegic solution containing various concentrations of ATP. Infusion was then terminated and the entire heart was maintained in a state of normothermic (37° C) ischemic arrest for 40 minutes. After this period, the heart was reperfused initially in the Langendorff mode for 15 minutes with collection of coronary effluent for creatine kinase (CK) determination." During this period, the time elapsed before the resumption of regular heart rhythm was noted. If regular rhythm had not resumed by the end of the initial 15 minute reperfusion period, the hearts were electrically defibrillated. After the 15 minute reperfusion period, the hearts were converted to the working mode for a further 20 minutes and the recovery of cardiac function was recorded. At the end of each experiment, hearts were heated to 110° C for 24 hours for the determination of dry weight. Hypothermia studies. The same protocol (Fig. 1) was followed, except that the ischemic coronary infusion of the cardioplegic solution was performed at 20° C and the heart was maintained for 270 minutes of hypothermic (20° C) ischemia. Cardioplegic solution at 20° C was reinfused for 3 minutes every 30 minutes during the

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Fig. 2. Adenosine triphosphate (ATP): Normothermic functional dose-response study. The relationship between the concentration of ATP (rnmol/L) in the cardioplegicsolution is compared to the postischemic recovery of (A) aortic flow and (B) minute work, both expressed as a percentage of their preischemic control values. Recoverywas measured at the end of a 35 minute reperfusion period after 40 minutes of normothermic (37 C) ischemic cardiac arrest. Each point represents a mean of at least six hearts and the bars indicate the standard error of the mean. Statistical significance compared to the ATP-free control groups: *p < 0.05; **p < 0.02; ***p < 0.001; NS = not significant. 0

418

The Journal of Thoracic and Cardiovascular

Robinson. Braimbridge, Hearse

Surgery

Table n. The effect of adenosine triphosphate (ATPj concentration or other additives to the St Thomas' cardioplegic solution upon the postischemic recovery of various indices of cardiac function and upon enzyme leakage after 40 minutes of normothermic ischemia Aortic flow ATP concentration (mmol/L)

Control imlfmin)

Coronary flow

Percent recovery after 35 min reperjusion

Control imllmin]

Percent recovery after 35 min reperjusion

Cardiac output Control (mlfmin}

Percent recovery after 35 min reperfusion

o (control) 0.05 0.1 0.25 0.5 1.0 10.0*

67.1 67.8 67.6 72.7 73.7 62.5 61.6

Adenosine 0.1 mmol/L tlnorganic phosphate 0.1 rnmol/L

68.3 ± 3.6

40.3 ± 5.3'

25.0 ± 1.3

72.9 ± 2.2

93.3 ± 4.3

49.1 ± 3.5'

77.0 ± 1.9

33.6 ± 7.5

28.7 ± 1.0

73.2 ± 5.3

105.7 ± 1.8

44.5 ± 6.3

± 1.6 ± 1.8 ± 3.2 ± 2.4 ± 2.8 ± 3.1 ± 2.7

24.1 31.8 62.6 50.4 43.5 29.2 48.2

± 4.4

± 2.7 ± 4.7' ± 7.7' ± 6.\' ± 4.2 ± 7.4

24.5 26.8 26.7 28.0 27.0 26.0 28.6

± 0.7

± 1.5 ± 0.9 ± 0.6 ± 1.2 ± 1.5 ± 0.8

68.7 81.8 92.1 77.5 78.1 76.4 68.4

± 5.1 ± 5.0 ± 2.1' ± 2.9 ± 3.9 ± 5.6 ± 9.6

92.1 94.7 94.3 100.7 100.7 88.5 91.6

± 2.0 ± 7.8 ± 3.6 ± 2.7 ± 2.3 ± 3.4 ± 2.3

35.9 45.8 71.0 58.0 53.4 42.9 35.8

± 4.1 ± 2.7 ± 3.0' ± 6.3' ± 4.5' ± 3.8 ± 9.4

Statistical analysis: Percent recovery for each concentration group for each variable has been compared (Student's t test) with the control cardioplegic solution (no additives). Values for each concentration group are the mean of at least six hearts ± SEM. Only statistically significant values are marked. a = p < 0.05. b = P <0.02. c=p
270 minute ischemic period. The test cardioplegic solutions used during the hypothermic series were one of the following four types: (1) the base solution; (2) base solution plus the optimal concentration of ATP determined in the initial normothermic series; (3) base solution plus CP 10 mmoljL (its optimal concentration found in a previous study"); or (4) base solution plus both ATP and CP at their individual optimal dosages. ATP stability studies. Samples of cardioplegic solution containing ATP 2.0 mmoljL were prepared and maintained at either 37° or 20° C for various times. Aliquots of the two solutions were assayed at 0.5, 1,2,3, 4, 5,6, 7, and 8 hours for ATP content" to study the stability of this compound in solution. Expression of results. During the preischemic working control period, the following variables were recorded directly: heart rate, coronary flow, aortic flow, aortic pressure, and dP/dt. Cardiac output was derived from the sum of aortic and coronary flows, and the stroke volume was obtained by dividing cardiac output by heart rate. Minute work was derived by multiplying cardiac output by systolic pressure. During the working recovery period, the absolute values for the various indices of cardiac function in individual hearts were compared and expressed in terms of percent of those values obtained during the preischemic control period. In addition to

eliminating any inherent variability between individual hearts, this allowed the recovery of each index of function to be expressed as a percentage and to be related to the temperature or duration of ischemia and to the nature and concentration of any cardioplegic additive. CK leakage was expressed as international units per 15 minutes per gram dry weight. At least six hearts were used for each condition studied and all data were expressed as the mean ± standard error. Statistical analysis of the results was made by paired or unpaired Student's t test (whichever was appropriate) and statistical significance was assumed when p values were 0.05 or less. Results Normothermic dose-response curves for ATP protection. The results are shown in Table II and Figs. 2 and 3. Lower concentrations of ATP (0.1 to 0.5 mmoljL) afforded substantial additional protection, with peak protection occurring with 0.1 mmol/L. At this optimum, postischemic recovery for aortic flow was 62.6% ± 4.7% and for cardiac output, 71.0% ± 3.0%; these values were two to three times greater (p < 0.001) than the ATP-free control values (24.1% ± 4.4% and 35.9% ± 4.1%, respectively). This substantial improvement in protection was also reflected by other indicesof

Volume 93 Number 3 March 1987

Myocardial protection with HEP 419

Stroke volume Control (mlfbeat] 0.32 0.32 0.33 0.34 0.35 0.34 0.31

om

± ± 0.02 ± 0.01 ± ± ± 0.02

om om

±

om

Percent recovery after 35 min reperfusion 38.3 48.4 73.9 60.1 55.2 44.6 38.2

± 4.5 ± 2.1 ± 4.3d ± 5.7' ± 5.6' ± 4.0 ± 9.6

Minute work

dPldt Control (cm H;zOlsec) 5245.4 4368.0 4397.7 4576.0 4333.3 5162.3 5498.6

± 393.8 ± 398.2 ± 388.3 ± 120.1 ± 164.8 ± 250.6 ± 474.9

Percent recovery after 35 min

reperfusion 44.3 47.8 63.6 60.6 60.2 62.3 42.2

± 3.6 ± 2.8 ± 4.8' ± 6.0' ± 4.4b ± 4.6' ± 6.8

Control (1()5 dyne-em/mint 203.5 218.8 204.8 216.5 217.9 197.5 201.0

Percent recovery after 35 min reperfusion 26.6 34.0 61.3 46.5 43.1 32.4 25.2

± ± ± ±

8.9 15.5 16.2 9.5 ± 5.9 ± 6.6 ± 14.0

± 4.1 ± 1.8 ± 6.3 d ± 6.8' ± 5.4' ± 3.2 ± 8.1

Creatine kinase leakage (lUll5 minfgm dry weight) 80.4 71.4 49.4 58.1 90.3 74.6 77.8

± ± ± ±

9.3 10.1 4.3' 10.9 ± 11.6 ± 13.2 ± 7.8

0.34 ± 0.01

52.8 ± 3.4'

3986.7 ± 155.8

56.0 ± 3.1'

195.0 ± 13.0

38.8 ± 3.5'

65.8 ± 13.8

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45.8 ± 7.8

4298.7 ± 158.1

57.2 ± 7.5

229.7 ± 10.2

31.8 ± 5.8

67.7 ± 7.0

0.36 ±

cardiac function (see Table II). The dose-response curves for function were paralleled by the results for CK leakage (Table II and Fig. 3), again indicating substantial additional protection, which resulted in an almost 40% reduction (p < 0.01) of CK leakage at the optimal ATP concentration (0.1 mmoljL). The dose-responsecharacteristics for ATP conform to a bell-shaped curve (Fig. 2), with no significant beneficial effect in terms of function or enzyme leakage at high or low concentrations. The highest ATP concentration (l0.0 mmol/L) tested had a deleterious effect on postischemic recovery, only three of seven hearts recovering coordinated contractile activity. Hypothermic protection studies with ATP. The optimal ATP concentration of 0.1 mmoljL, determined in the normothermic studies, was used in studies involving 270 minutes of hypothermic ischemia (20 0 C). This duration of ischemia was selected because it produced a level of damage in control hearts sufficient to ensure only a 25% to 35% recovery of pump function. Any protection afforded by the addition of ATP could then be readily detected and measured. Fig. 4 shows that under conditions of extended hypothermic arrest, ATP affords substantial protection. There was a doubling of recovery of aortic flow (from 26.8% ± 8.4% in the control group to 58.0% ± 4.7% in the ATP group, p < 0.1); similar, significant, and substantial improvement was seen in minute work, stroke volume,cardiac output, dP/ dt, coronary flow,and aortic pressure. There was no significant change in heart rate. CK leakage was significantly lowered from 66.8 ± 11.9 1U/15 min/gm dry weight in the control group to

32.6 ± 3.3 1U/15 min/gm dry weight in the ATP group (p < 0.02). Hypothermic protection with CP alone. Additional studies with CP alone added to the cardioplegic solution at its optimal concentration (l0.0 mmol/L, determined in a previous study") were also performed during 270 minutes of hypothermic (20 C) ischemia. The results were similar in all respects to the ATP studies: Recovery of aortic flow was improved from 26.8% ± 8.4% in the control group to 57.9% ± 4.3% (p < 0.01) in CP group. Similar, significant improvement was observed for recovery of minute work, stroke volume, cardiac output, dP /dt, and aortic pressure. There was no significant difference in heart rate, coronary flow, or CK leakage when compared with control. Hypothermic protection studies with ATP and CP combined. A final series of studies was performed with a cardioplegic solution containing optimal concentrations of both ATP (0.1 mmoljL) and CP (10.0 mmol/L), Again, these studies were performed with 270 minutes of hypothermic (20 C) ischemia. Substantial improvements in the recovery of aortic flow were observed: from 26.8% ± 8.4% in the control group to 79.7% ± 1.1% in the treated group (p < 0.001). Significant improvements in minute work, stroke volume, cardiac output, dP/ dt, coronary flow, and aortic pressure also occurred, and CK leakage was decreased from 66.8 ± 11.9 to 26.1 ± 8.2 1U/15 min/gm dry weight (p < 0.02). When all four hypothermia study groups were compared (Fig. 5), it was clear that either ATP or CP alone at its optimal concentration markedly enhanced the protective properties of the St. Thomas' Hospital cardio0

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The Journal of

420 Robinson, Braimbridge, Hearse

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plegic solution. When included together, an additive effect provided even greater protection than with either substance alone. Recovery of cardiac output, for example, improved from 33.4% ± 6.3% in the control group to 64.4% ± 3.4% in the ATP group (p < 0.01), to 60.3% ± 3.2% in the CP group (p < 0.01), but to 80.7% ± 0.9% in the ATP plus CP group (p < 0.001 compared with each of the three other groups). Antiarrhythmic effects of ATP. During the normothermic studies we recorded the number of hearts requiring electrical defibrillation during reperfusion and the duration of reperfusion that elapsed before there was a return to normal rhythm. ATP added to the cardioplegic solution in the lower concentration ranges (0.05, 0.1, and 0.5 mmol/L) abolished the need for defibrillation; by contrast, 30% of hearts in the control group and 43% in the high ATP concentration group (1.0 and 10.0 mmol/L, respectively) required defibrillation. Furthermore, ATP at its optimal concentration of 0.1 mmol/L significantly reduced the time required for return to regular rhythm from 7.9 ± 2.0 minutes in the control group to 3.5 ± 0.4 minutes in the ATP group (p < 0.05). In the hypothermic studies, all hearts in all groups spontaneously defibrillated. Both ATP and CP addition tended to reduce the mean time to return of regular

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Fig. 4. Protective effects of ATP during prolonged hypothermic ischemia. The postischemic recovery of various functional indices (expressed as a percent of the preischemic baseline control value) is compared in control hearts (open columns) receiving ATP-free cardioplegic solution and in treated hearts receiving cardioplegic solution containing an ATP concentration of 0.1 mmoljL (shaded columns). Recovery was measured at the end of a 35 minute reperfusion period after 270 minutes of hypothermic (20 0 C) ischemia. Each column represents the mean of six hearts and bars represent the standard error of the mean. Statistical significance compared to the ATP-free control group: *p < 0.01; **p < 0.001.

rhythm (3.8 ± 1.7 minutes in the control group, 0.6 ± 0.1 minutes in the ATP group, 0.7 ± 0.06 minutes in the CP group, and 0.5 ± 0.06 minutes in the CP plus ATP group). However, none of these changes achieved statistical significance. Stability of ATP in the St. Thomas's Hospital cardioplegic solution. ATP is a labile compound and its use in any cardioplegic solution requires that it remain structurally intact. To assess stability, we kept two cardioplegic solutions containing ATP at a calculated concentration of 2.0 mmol/L at 20 and 37° C for 8 hours. Chemical assay at the onset of the experiment confirmed the ATP content. At all times throughout the 8 hours studied in both groups, the ATP concentration remained unchanged (2.00 mmol/L in the 20° C storage group and 2.04 mmoljL in the 37° C storage group). Studies of possible mechanisms of action for ATP protection. Here we investigated whether ATP-induced protection may be due to its degradation products or to its ability to influence coronary vascular tone. Adenosine and inorganic phosphate. To establish that 0

Volume 93 Number 3 March 1987

the protective effects of ATP were attributable to the intact molecule, rather than to its degradation products, we added adenosine (0.1 mmol/L) or inorganic phosphate (0.1 mmoljL) to the cardioplegic solution and subjected the hearts to 40 minutes of normothermic ischemia. As indicated in Table II, adenosine addition afforded a small but significant improvement in functional recovery (p < 0.05) but failed to reduce CK leakage. The ATP concentration of 0.1 mmoljL, however, afforded significantly better myocardial protection than adenosine for all indices studied except dP/dt and aortic pressure. The addition of 0.1 mmol/L trisodium orthophosphate (inorganic phosphate) to the cardioplegic solution had no significant positive effect on function or CK leakage. In fact, inorganic phosphate was deleterious to postischemic function, as one third of the hearts in this group could not be defibrillated. The adverse effects of inorganic phosphate addition may be mediated through its interaction with calcium, leading to the formation of insoluble calcium phosphate precipitates. ATP and coronary vascular resistance. In this isolated rat heart preparation coronary perfusion pressure is constant,· being set at 60 em H 20 pressure during cardioplegic infusion or at 100 ern H 20 pressure during aerobic Langendorff perfusion. Any increase in coronary flow reflects a proportional decrease in coronary vascular resistance and vice versa. In the normothermia series, the infused volume of cardioplegic solution (3 minutes of gravity flow at 60 em H 20 of pressure) was measured. There was a significant increase in infused volume with a fall in coronary vascular resistance in the low concentration ATP groups (0.05, 0.1, and 0.25 mmoljL) when compared to the control group (45.9 ± 1.5 mI for the 0.1 mmoljL ATP group versus 37.4 ± 1.1 mI for the control group, p < 0.001). The opposite occurred in the high ATP concentration groups (1.0 and 10.0 mmol/L), in which there was a significant decrease in volume of infused cardioplegic solution (37.4 ± 1.1 mI for control versus 10.9 ± 1.7 mI in the 10.0 mmoljL ATP group, p < 0.001). Three of seven hearts in the 10.0 mmol/L ATP group continued to beat for over 2 minutes during the infusion of the ATP-containing cardioplegic solution, probably because of poor distribution of solution as a consequence of high coronary vascular resistance. In all other test groups, infusion of the cardioplegic solution resulted in almost instantaneous diastolic arrest. There were no significant changes in coronary flow and resistance during cardioplegic infusion in the adenosine or inorganic phosphate study groups. During postischemic reperfusion (gravity flow at 100

Myocardial protection with HEP

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Fig. 5. Protective effects of ATP, CP, or ATP plus CP during prolonged hypothermic ischemia. The postischemic recovery of various functional indices (expressed as a percent of the preischemic baseline control value) is compared in control hearts receiving ATP-free cardioplegic solution, hearts treated with solution containing ATP 0.1 mmoljL, solution containing CP 10.0 mrnol/L, and solution containing ATP 0.1 mrnol/L and CP 10.0 mmol/L, Recovery was measured at the end of a 35 minute reperfusion period after 270 minutes of hypothermic (20 C) ischemia. Each column represents the mean of six hearts and the bars represent the standard error of the mean. Statistical comparisons are described as follows: *p < 0.01 compared to control; **p < 0.01 compared to ATP alone and p < 0.001 compared to CP alone; tp < 0.001 compared to ATP or CP alone. 0

em H 20 pressure), there were significant increases in coronary flow (decreased coronary vascular resistance) in the low-concentration ATP groups (0.05, 0.1 and 0.25 mmol/L) with total coronary flow over 15 minutes being 265.9 ± 5.7 mI in the 0.1 mmoljL ATP group compared with 198.0 ± 4.5 mI in the control group (p < 0.001). The previous effect on cardioplegic infusion noted in the 1.0 and 10.0 ml/L ATP groups was not observed during the reperfusion phase. There were no significant changes during reperfusion in the free adenosine or inorganic phosphate groups.

Discussion Myocardial ischemia initiates a sequence of cellular changes that, unless checked, culminate in irreversible

422 Robinson. Braimbridge, Hearse

injury and cell death. Initially, mitochondrial oxidative phosphorylation is severely reduced, and soon ATP and CP levels decline. Despite cessation of contractile activity from oxygen and energy lack, cellular metabolism continues, contributing to further ATP and CP depletion.':3, 7, 42, 43 This energy depletion appears to play a vital role in determining cell viability and in particular determining whether contractile function resumes on reperfusion. 1,3,44-46 Cardioplegic solutions conserve HEP by hypothermia and by inducing rapid diastolic arrest. However, neither potassium arrest nor hypothermia completely halts cellular energy utilization, and cytoplasmic energy stores continue to dwindle." 47 Attempts to combat HEP depletion by manipulating adenine nucleotide synthetic pathways have included the use of various substrates: ribose," glucose-insulin-potassium,9,16 acetate," pentose," inosine," creatine," L-glutamate," and 5-aminoimidazol-4-carboxamide riboside (AICAR).14,17 Another approach involves the use of metabolic inhibitors such as erythro-9-adenine hydrochloride (adenosine deaminase inhibitor) plus adenosine," allopurinol, II, 12 dipyridamole, 10, 22 propranolol, 19,22 and a ,8-methyleneadenosine 5' diphosphate (5' nucleotidase inhibitor)." A third method has been to adminiter HEP itself, and uniformly beneficial effects have been reported with exogenous ATP or CP. 23-29,48-61 Some studies have claimed direct uptake of HEP in its intact molecular form into cells of the myocardium and other tissues." 51, 55, 57, 61 The present study defined a bell-shaped dose-response curve for ATP with an optimal protective concentration of 0.1 mmoljL. Higher or lower concentrations protected less; the highest ATP concentration tested even had a detrimental effect, with an accompanying increase in coronary vascular resistance. High ATP concentrations may depress recovery of postischemic function by causing poor myocardial distribution of cardioplegic solution or by the known calcium-chelating effect of high ATP concentrations. High concentrations of extracellular ATP infused suddenly may exert some profound effect on the handling of calcium by the myocardium, as seen in the calcium paradox." Our evidence for improved cardiac protection is, interestingly, supported by Parratt and Marshall," who found protective effects of exogenous ATP on the response of isolated guinea pig atrial muscle to acute anoxia (optimal ATP concentration 0.118 mmoljL). Fedelesova and associates" evaluated the effect of intracoronary ATP injection in cold solution on myocardial adenine nucleotides in the isolated ischemic dog heart; they found slowing of glycogen and HEP depletion, at a dose similar to that used in the present study.

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Siska and associates" also demonstrated that intracoronary ATP injection (4 to 6 J,Lmolj gm myocardium) protected in vivo the hypothermic (24 0 C) dog heart during ischemia. The relatively.narrow ATP dose-response curve for protection may account for the failure of other investigators to demonstrate beneficial effects. Brown, Mulder, and Chiu" studied intact dogs exposed to 2 hours of hypothermic (25 0 C) ischemia and found that the use of a cardioplegic solution containing an ATP concentration of 3 to 50 mmoljL resulted in poor hemodynamic performance during reperfusion, with the worst results (no recovery) observed with the higher ATP concentrations. Reibel and Rovetto? found that an ATP concentration of 0.5 mmoljL did not enhance postischemic recovery in isolated rat hearts. The concentrations of ATP used in both studies fall outside the protective range of the dose-response curve. The biochemical dogma that energy-rich phosphates do not cross the cell membrane suggests that the observed protective effects of ATP are due to degradation products. Although adenosine exerted a small but significant protective effect in our studies, the extent of this protection was much less than that seen with ATP. Despite the vasodilatory effect of the low concentrations of ATP during cardioplegic infusion, adenosine at the same concentration exerted no such effect. This is not unexpected because ATP is known to be four times more potent than adenosine." However, the fact that adenosine exerted any protective effect suggests that part of the action of ATP might be attributed to adenosine released by ATP hydrolysis during its stasis in the heart. Since adenosine is freely transported across the cell membrane, it may diffuse into the cell and help replenish that lost as a consequence of intracellular ATP degradation. Foker, Einzig, and Wang" found no effect on myocardial ATP of intracoronary adenosine infusion in the in vivo dog after 20 minutes of normothermic ischemia, although this may have been due to coronary blood adenosine deaminase, the enzyme that degrades adenosine to inosine. Reibel and Rovetto? also found no protective effect of adenosine in the ischemic rat heart. Inorganic phosphate added to cardioplegic solution afforded no protection; in fact, a detrimental effect on postischemic functional recovery was observed. Parratt and Marshall" also found that inorganic phosphate accentuated the negative inotropic effect of anoxia in the isolated guinea pig atrium. We might conclude that the intact ATP molecule, at its optimal concentration, is required for maximal myocardial protection. Clinical cardiac operations are performed with the myocardium below 20 0 C. This study's protocol also

Volume 93 Number 3 March 1987

involved testing additives during extended periods of hypothermic ischemia. ATP at its optimal concentration provided good myocardial protection over 4lh hours, an effect similar to that afforded by CP at its optimal dosage. 30 However, ATP and CP together at their individual optimal concentrations provided significantly better myocardial protection than either substance alone. One of the important drawbacks of cold cardioplegic myocardial protection is the occurrence of postischemic arrhythmias. In this study, ATP significantly reduced arrhythmias after normothermic and hypothermic ischemia. It obviated the need for electrical defibrillation and reduced the time that elapsed before return to regular rhythm. Recent laboratory and clinical studies have shown antiarrhythmic actions of ATP, by slowing atrioventricular node conduction and for terminating supraventricular tachyarrhythmias in children.v" CP has also previously demonstrated similar antiarrhythmic properties." It is not known whether this effect of ATP was indirect by improved myocardial protection or whether it was a specific mechanism, such as suggested for CP.67 Prompt resumption of sinus rhythm after cardioplegic arrest would represent a major clinical improvement. In a consideration of the mechanisms underlying the effects of ATP and CP, a number of questions arise: (1) Do they act at an extracellular or intracellular level? (2) If at an intracellular level, how is access gained to the cytoplasmic space? (3) What metabolic processes are protected? (4) Do they have a common mechanism? However, an understanding of the normal myocyte interactions of HEP is essential first to propose mechanisms for the positive effects of exogenous ATP and CP during ischemia. The most attractive theory of physiologic intracellular energy supply and transport involves the concept of functional compartmentalization of ATP at contractile, membrane, and mitochondrial sites, with these relatively immobile pools being connected by the effector CP shuttle,"?" Various studies have also demonstrated active cell membrane enzyme systems such as the MM isoenzyme of CK71 and membrane Na-K-ATPase,72.73 which might allow significant interaction with exogenous HEP. It is generally believed that ATP cannot penetrate the cell membrane intact because of the polar nature of nucleotides, their instability in solution (although not seen in the present study), their high reactivity and electrical charge, and their large size. However, effects observed with exogenous ATP and CP in the ischemic myocardium can only be explained by proposing either an intracellular site of action or at least an effect at the

Myocardial protection with HEP 42 3

level of the cell membrane. Chaudry and Gould" demonstrated uptake of ATP by in vitro soleus muscle, as did Hoffman and Okita" in myocardium and Chaudry, Sayeed, and Baue" in liver and kidney. Rosenshtraukh and associates" showed direct entry of CP into isolated frog heart, raising intracellular CP content and increasing contractile force, although this effect has not been demonstrated in mammals. Fedelesova and assoelates," however, suggested that ATP is dephosphorylated at the cell membrane, perhaps by one of the known membrane ATPases,72-74 and in some manner replenishes the intracellular adenosine nucleotide pool. The similar protection achieved by the optimal concentrations of both ATP and CP suggests a common mechanism. Chaudry and Baue" recently reviewed the use of HEP during ischemia, including the successful use of ATP in treating shock and in postischemic renal and hepatic injury. Such reports suggest that the protective properties of CP may be mediated by ATP. Such an argument is strengthened by our observation that at least one third of the infused CP disappears from the vascular space during its stasis in the heart. 30 This could be the result of hydrolysis, extracellular binding, or intracellular uptake, but it is conceivable that CP acts a~ an HEP source for rephosphorylation of adenosine diphosphate at some key cellular site. Such a situation might explain why greater quantities of CP are required to achieve a protective effect similar to ATP. Because even during ischemia intracellular HEP content is in the millimolar range, significant supplementation of reserves would require that extracellular ATP and CP would also have to be in the millimolar range. Although this is true for CP, ATP exerts its optimal protection at micromolar concentrations and this argues against a common intracellular site of action. Proposing a common site of action is also challenged by our observation that CP and ATP exert an additive protective effect, providing strong evidence for a different mechanism of action. ATP may instead act as an energy source for a small but important cellular process (possibly at the cell membrane), which plays a critical role in irreversible cell injury. An alternative explanation might involve multiple sites of action some of which are shared by both compounds. An extracellular site of action for ATP may be postulated because extracellular ATP can be detected under normal conditions, particularly during hypoxia." ATP may act as a substrate source for extracellular or membrane-bound energy-requiring enzyme systems,72.74 which are vulnerable to energy deprivation during ischemia. Extracellular ATP (or CP) might then protect membrane-bound ATP-dependent processes by ensur-

ing a continuous energy supply.

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424 Robinson, Braimbridge, Hearse

Exogenous ATP is known to protect other organs from ischemia, as reported by Andrews and Coffey," who protected rat kidneys from ischemic failure by ATP-MgCI 2 pretreatment. Garvin and associates" also found 2.5 mmoljL ATP- MgCl 2 pretreatment maintained intracellular nucleotide levels and protected canine kidneys under hypothermic storage for 24 hours. Ohkawa, Clemens, and Chaundry" reported improved survival with infusion of ATP-MgCI2 into rats after 90 minutes of hepatic ischemia. Kraynack, Gintautas, and Hinshaw" demonstrated that intracerebroventricular pretreatment of rats with ATP exposed to 60 minutes of hypoxia increased survival by 250%. Finally, Chaundry and Baue" reported favorable results in animal models in which ATP-MgCI 2 was used for the treatment of shock and postischemic renal and hepatic injury. More relevant to the present study, McDonagh and associates" found in dogs that continuous intracoronary ATP-MgCI 2 infusion (0.04 mmoljL) during postischemic reperfusion improved the return of cardiac function, but the effect was less with high-dose ATP (1.0 mmoljL). Dissimilar responses for low-dose and high-dose ATP treatment were found in our study and by Clemens and Forrester" with isolated rat hearts. Clinical application of systemic ATP has taken several forms. Happert, Pilardeau, and Catel" reported success using oral ATP for various cardiac arrhythmias. Greco and colleagues" found intravenous ATP effective in terminating supraventricular tachycardia in 90% of infants. Despite the promise suggested by these preliminary clinical studies, more definitive controlled trials will be needed to determine whether ATP offers significant advantages over conventional cardiac treatment. Concluding comments The findings from the present study combined with our previous study on Cp30 suggest that exogenous ATP and CP used at their optimal dosage each offer considerable myocardial protection during prolonged hypothermic ischemia as manifested by improved functional recovery, decreased CK leakage, and reduction of arrhythmias. When combined, ATP and CP act synergistically to provide even greater protection than either alone. As these two compounds appear to offer advantages to the normal hearts used in this study, they may provide even greater benefit in hypertrophied hearts that have diminished subendocardial ATP stores, a factor that probably contributes to their enhanced sensitivity to ischemia." Although these results appear interesting, an element of caution should be raised with regard to the clinical use of ATP, CP, or both in cardioplegic solutions. Further work is still warranted before these

promising cardioplegic additives can be recommended for use during cardiac operations. The assistance of Mrs. C. Erlebach and Ms. S. Langston is gratefully acknowledged. REFERENCES I. Jennings RB, Hawkins HK, Lowe JE, Hill ML, Klotman S, Reimer KA. Relation between high energy phosphate and lethal injury in myocardial ischemia in the dog. Am J Pathol 1978;92:187-207. 2. Hearse DJ, Braimbridge MV, Jynge P. Ischemia and reperfusion: the progression and prevention of tissue injury. chap. 2. In: Protection of the ischemic myocardium: cardioplegia. 1st ed. New York: Raven Press, 1981:21-49. 3. Reimer KA' Hill ML, Jennings RB. Prolonged depletion of ATP because of delayed repletion of the adenine nucleotide pool following reversible myocardial ischemic injury in dogs. Adv Myocardiol 1983;4:395-407. 4. Hearse DJ, Braimbridge MV, Jynge P: Methods of assessment and results of cardiac surgery using cardioplegia. chap. 12. In: Protection of the ischemic myocardium: cardioplegia. 1st ed. New York: Raven Press, 1981:375-410. 5. Hearse DJ, Steward DA, Chain EB. Recovery from cardiac bypass and elective cardiac arrest. Circ Res 1974;35:448-57. 6. Vary TC, Angelakos ET, Schaffer SW. Relationship between adenine nucleotide metabolism and irreversible ischemic tissue damage in isolated perfused rat heart. Circ Res 1979;45:218-25. 7. Reibel DK, Rovetto MJ. Myocardial ATP synthesis and mechanical function following oxygen deficiency. Am J Physiol I 978;234:H620-4. 8. Pasque MK, Spray TL, Pellom GL, et al. Riboseenhanced myocardial recovery following ischemia in the isolated working rat heart. J THORAC CARDIOVASC SURG 1984;83:390-8. 9. Levitsky S, Feinberg H. Protection of the myocardium with high-energy solutions. Ann Thorac Surg 1975;20:8690. 10. Degenring FH, Curnish RR, Rubio R, Berne RM. Effect of dipyridamole on myocardial adenosine metabolism and coronary flow in hypoxia and reactive hyperemia in the isolated perfused guinea pig heart. J Mol Cell Cardiol 1976;8:877-88. 11. Lindsay WG, Toledo-Pereyra LH, Foker JE, Varco RL. Metabolic myocardial protection with allopurinol during cardiopulmonary bypass and aortic cross-clamping. Surg Forum 1975;26:259-60. 12. DeWall RA, Vasko KA' Stanley EL, Kezdi P. Responses of the ischemic myocardium to allopurinol. Am Heart J 1971;82:362-70. 13. Foker JE, Einzig S, Wang T. Adenosine metabolism and myocardial preservation. J THORAC CARDIOVASC SURG 1980;80:506-16. 14. Swain JL, Hines JJ, Sabina RL, Holmes EW. Accelerated repletion of ATP and GTP pools in postischemic

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nism of glycolytic impairment after adenosine triphosphate depletion in the perfused rat heart. J Physiol 1980; 301:337-47. Hearse DJ, Braimbridge MV, Jynge P: Comparison of methods. chap. 4. In: Protection of the ischemic myocardium: cardioplegia. 1st ed. New York: Raven Press, 1981:95-147. Marshall JM, Andrus EC. Comparison of effects of various phosphate compounds and aluminum silicate on isolated frog heart. Proc Soc Exp BioI Med 1953;82:22831. Hoffman PC, Okita GT. Penetration of ATP into the myocardium. Proc Soc Exp BioI Med 1965;119:573-6. Chaundry IH, Gould MK. Effect of externally-added ATP on glucose uptake by isolated rat soleus muscle. Biochim Biophys Acta 1970;196:327-35. Chaundry m, Gould MK. Evidence for the uptake of ATP by rat soleus muscle in vitro. Biochim Biophys Acat 1970;196:320-6. Ziegelhoffer A, Fedelesova M, Kostolansky S. Specific ATP action on metabolism of isolated heart: influence of pH, divalent cation concentration and stability of complexes. Acta BioI Med Germ 1972;28:893-900. Wilkinson JH, Robinson JM. Effect of ATP on release of intracellular enzymes from damaged cells. Nature 1974; 249(5458):662-3. Wilkinson JH, Robinson JM. Effect of energy-rich compounds on release of intracellular enzymes from human leukocytes and rat lymphocytes. Clin Chern 1974; 20:1331-6. Chaundry IH, Sayeed MM, Baue AE. Uptake of ATP by liver and kidney in vitro. Can J Physiol Pharmacol 1976;54:742-9. Vassort G, Ventura-Clapier R. Significance of creatine phosphate on the hypodynamic frog heart. J Physiol (London) 1977;269:86-7. Chaudry IH, Sayeed MM, Baue AE. Evidence for enhanced uptake of ATP by. liver and kidney in hemorrhagic shock. Am J Physiol 1977;233:R83-8. Rosenshtraukh LV, Saks VA, Yuriavichus lA, et al. Effect of creatine phosphate on the slow inward calcium current, action potential, and the contractile force of frog atriurri and ventricle. Biochem Med 1979;21:1-15. Vacca C; Giordano L, Imperatore A, et al: [Sodium phosphocreatine, hemodynamics; and cardiac metabolism: experimental research.] Boll Chim Farm 1980; 119:429-33 (Ital.). F1itney FW, Singh J. Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides. J Physiol (London) 1980; 304:21-42. Rosenshtraukh LV, Saks VA, Undrovinas AI, Chazov EI, Smirnov VN, Sharov VG. Studies of energy transport in heart cells: The effect of creatine phosphate on the frog ventricular contractile force and action potential duration. Biochem Med 1978;19:148-64.

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62. Clemens MG. Role of extracellular adenosine triphosphate during hypoxia and calcium deprivation in the isolated working rat heart. Diss Abst Int 1981 ;41:2499500B. 63. Brown PR, Mulder DS, Chiu RC. Effects of hypothermic K-cardioplegia and ATP- Mg solution of myocardial ATP content and function. Surg Forum 1979;30:248-50. 64. Winbury MM, Papierski D, Hemmer, M, Hamburger W. Coronary dilator action of adenine-ATP series. J Pharmacol Exp Ther 1953;109:225-61. 65. Greco R, Musto B, Arienzo V, Alborino A, Garafalo S, Marsico F. Treatment of paroxysmal supraventricular tachycardia in infancy with digitalis, adenosine-5'triphosphate, and verapamil: a comparative study. Circulation 1982;66:504-8. 66. Belardinelli L, Shryock J, West GA, Clemo HF, DiMarco JP, Berne RM. Effects of adenosine and adenosine nucleotides on the atrioventricular node of isolated guinea pig hearts. Circulation 1984;70:1083-91. 67. Fagbemi 0, Kane KA, Parratt JR. Creatine phosphate suppresses ventricular arrhythmias resulting from coronary legitation. J Cardiovascular Pharmacol 1982;4:538. 68. Gudbjarnason S, Mathes P, Ravens KG. Functional compartmentation of ATP and creatine phosphate in heart muscle. J Mol Cell Cardiol 1970;1:325-39. 69. Bessman SP, Geiger PJ. Transport of energy in muscle: the phosphoryl-creatine shuttle. Science 1981;211 :44852. 70. Saks VA, Rosenshtraukh IV, Smirnov VN, Chazov EL. Role of creatine phosphokinase in cellular function and metabolism. Can J Physiol Pharmacol 1978;56: 691-706. 71. Saks VA, Lipina NV, Sharov VG, Smirnov VN, Chazov E, Grosse R. The localization of the MM isoenzyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabaininhibited Na+,K+-ATPase. Biochim Biophys Acta 1977; 465:550-8. 72. Agren G, Poten J, Ronquist G, Westermark B. Demonstration of an ATPase at the cell surface of intact normal and neoplastic human cells in culture. J Cell Physiol 1971;78:171-6. 73. Proverbio F, Hoffman JF. Membrane compartmentalized ATP and its preferential use by the NA,K-ATPase of human red cell ghosts. J Gen Physiol 1977;69:605-32. 74. Williamson JR, Dipietro DL. Evidence for extracellular enzymatic activity of the isolated perfused rat heart. Biochem J 1965;95:226-32. 75. Clemens MG, Forrester T. Appearance of adenosine triphosphate in the coronary sinus effluent from isolated working rat heart in response to hypoxia. J Physiol 1980; 312:143-58. 76. Andrews PM, Coffey AK. Protection of kidneys from acute renal failure resulting from normothermic ischemia. Lab Invest 1983;49:87-98.

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77. Garvin PJ, Carney K, Jellinek M, Niehoff M, Casteneda M, Codd JE. The effects of ATP-MgCI 2 and dipyridamole in cold-storage preservation. J Surg Res 1983; 34:443-50. 78. Kraynack BJ, Gintautas J, Hinshaw J, Adenosine triphosphate increases survival time during hypoxia. Neuropharmacology 1981;20:887-90. 79. Clemens MG, Forrester T. Exacerbation of the calcium paradox with exogenous ATP in isolated working rat heart. Q J Exp Physiol 1982;67:655-62.

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80. Happert JL, Pilardeau P, Catel P. [Clinical experiments with Striadyne Forte (ATP) on the digitised and nondigitised senile heart.] Gazette Med Fr 1977;84:2654-56 (Fr.). 81. Peyton RB, Jones RN, Attarian D, et al. Depressed high-energy phosphate content in hypertrophied ventricles of animals and man. Ann Surg 1982;196:278-84.

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