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Ann Thorac Surg 2000;70:756-764
© 2000 The Society of Thoracic Surgeons

Original articles: cardiovascular

The role of cardioplegia induction temperature and amino acid enrichment in neonatal myocardial protection

Michael T. Kronon, MDb, Bradley S. Allen, MDa, Kirk S. Bolling, MDb, Shaikh Rahman, PhDb, Tingrong Wang, MDb, Hersh S. Maniar, BSb, Sunil M. Prasad, BSb, Michel N. Ilbawi, MDa

a Division of Cardiovascular Surgery, The Heart Institute for Children, Hope Children’s Hospital, Oak Lawn, Illinois, USA
b The University of Illinois at Chicago, Chicago, Illinois, USA

Address reprint requests to Dr Allen, Heart Institute for Children, Hope Children’s Hospital, 4440 W 95th St, Oak Lawn, IL 60453


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Warm cardioplegic induction improves the ischemically "stressed" adult heart. However, it is rarely used in infants, despite the fact that many newborn hearts are stressed by other factors such as hypoxia. The need for amino acids as well as their mechanism of action has also not been studied.

Methods. We first assessed the role of cardioplegic induction temperature in 10 nonhypoxic neonatal piglets undergoing 70 minutes of multidose blood cardioplegic arrest. Five piglets (group 1) received a cold (4°C) induction, and 5 (group 2) a warm (37°C) induction. Twenty-six other piglets underwent ventilator hypoxia (fraction of inspired oxygen, 8% to 10%) for 60 minutes before cardiopulmonary bypass (stress). Six piglets (group 3) then underwent 70 minutes of cardiopulmonary bypass without ischemia (hypoxia controls), and 20 underwent 70 minutes of cardioplegic arrest. Five of these (group 4) received cold cardioplegic induction, and 15 received warm induction; in 5 of these (group 5), the warm cardioplegic solution contained amino acids, in 5 others (group 6), it was unsupplemented, and in the remaining 5 (group 7), nitroglycerin was added to determine the role of vasodilation. Myocardial function was assessed by pressure-volume loops (expressed as a percent of control), and coronary vascular resistance was measured with cardioplegic infusions.

Results. In nonhypoxic (normal) piglets, cold (group 1) and warm (group 2) induction completely preserved systolic function (end-systolic elastance, 100% versus 104%) and preload recruitable stroke work (100% versus 102%), with minimal increase in diastolic compliance (162% versus 156%). Hypoxia–reoxygenation alone (group 3) depressed systolic function (end-systolic elastance, 51% ± 2%) and preload recruitable stroke work (54% ± 3%), and raised diastolic stiffness (260% ± 15%). The detrimental effects of reoxygenation persisted (unchanged from reoxygenation alone) with cold induction (group 4) or warm induction without amino acids (groups 6 and 7). In contrast, warm induction with amino acids (group 5) restored systolic function (end-systolic elastance, 105% ± 3%; p < 0.001 versus groups 3, 4, 6, and 7) and preload recruitable stroke work (103% ± 2%; p < 0.001 versus groups 3, 4, 6, and 7), and decreased diastolic stiffness (154% ± 7%; p < 0.001 versus groups 3, 4, 6, and 7). However, there was no difference in myocardial oxygen consumption in hypoxic hearts receiving a warm induction (6.9 versus 6.5 versus 7.3 mL/g per 5 minutes) (groups 5, 6, 7), and coronary vascular resistance was lowest with nitroglycerin (group 7).

Conclusions. Cardioplegic induction can be given either warm or cold in nonhypoxic neonatal hearts. In contrast, only warm induction with amino acids repairs the hypoxic injury, but the primary mechanism of action is not related to increased metabolic activity or vasodilation.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Postoperative low cardiac output continues to be a major contributor to morbidity and mortality in pediatric cardiac operations because the neonatal heart has a reduced systolic and diastolic functional reserve, as well as a reduced response to inotropic agents compared with the adult [1–3]. Preservation of myocardial function in neonates during cardiac operations therefore assumes even greater importance, because a preoperative insult (stress) is less well tolerated, and more difficult to treat [1, 4–6]. In ischemically "stressed" adult hearts, a warm cardioplegic induction improves myocardial recovery, and function is further enhanced by enriching the warm cardioplegic solution with the amino acids aspartate (Asp) and glutamate (Glut) [7, 8]. Several mechanisms have been proposed to explain the action of amino acids, including primarily replenishing depleted Krebs cycle intermediates to enable the heart to increase adenosine triphosphate (ATP) production [8]. This hypothesis is supported by the high oxygen uptake observed in ischemic adult hearts when the warm cardioplegic solution is enriched with amino acids. In addition, associated vasodilation produced by amino acids reduces coronary vascular resistance (CVR), thus improving cardioplegic distribution [8, 9]. In the infant myocardium, myocardial energy stores are greater, and, unlike adult hearts, preoperative ischemia is uncommon as there is no coronary occlusion [1, 2, 10]. This has led to the perception that warm induction, as well as amino acid supplementation, is unnecessary [1, 2, 10]. Pediatric hearts are, however, often stressed by other factors such as hypoxia or pressure-volume overload, which although different from ischemia, can result in substrate and energy depletion [1, 2, 11–15]. Besides differences in the type of stress, extrapolation of adult cardioplegia strategies to the neonate is further complicated by the structural, functional, and metabolic differences between mature and immature hearts [3, 4]. This study uses a neonatal heart model that mimics the clinical setting to investigate the questions of cardioplegic induction temperature, amino acid enrichment, and the mechanism by which amino acids work. In contrast to adult studies, it subjects hearts to a hypoxic instead of an ischemic stress, because hypoxia is clinically more applicable to pediatric patients. First, the question of induction temperature is investigated in normal as well as stressed (hypoxic) neonatal hearts, because this is where warm induction may be most applicable. Second, the question of Asp or Glut enrichment of the induction solution is examined to determine whether amino acids confer additional benefit. And third, it attempts to define the mechanism of action of amino acids by using nitroglycerin (NTG) to investigate the role of vasodilation and measuring metabolic variables to examine enhanced substrate production.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Thirty-six neonatal (5- to 18-day-old) piglets (3.5 to 5 kg) were premedicated with 40 mg/kg ketamine intramuscularly and anesthetized with 30 mg/kg phenobarbital intraperitoneally, followed by 5 mg/kg intravenously each hour, and the lungs were ventilated by means of a tracheotomy using a volume ventilator (Servo 900B, Siemens/Elema, Solna, Sweden). All animals received humane care in compliance with the principles ("Principles of Laboratory Animal Care") formulated by the National Society for Medical Research, and "Guide for the Care and Use of Laboratory Animals," prepared by the National Academy of Sciences and published by the National Institutes of Health publication 96–03, revised 1996. The cardiopulmonary bypass (CPB) circuit was anticoagulated with heparin, primed with packed red blood cells from donor pigs, and made normocalcemic with CaC12. The experimental preparation, including cannulation for bypass and blood sample procurement, is comparable to that previously described [11, 16].

Cardioplegia protocol
Cardioplegic solutions (CAPS Service, Research Medical Inc, Salt Lake City, UT) are shown in Tables 1 and 2. Five minutes after initiating CPB, piglets underwent 70 minutes of cardioplegic arrest using a protocol consisting of 5 minutes of cardioplegic induction with substrate-enriched blood cardioplegic solution (Table 1), followed by 4 minutes of cold multidose cardioplegic solution (Table 2), a 2-minute multidose infusion every 20 minutes, and a 4-minute warm (37°C) substrate-enriched cardioplegic reperfusate ("hot shot") before aortic unclamping. Cardioplegic solution was always infused at a continuously measured aortic root pressure of 40 to 50 mm Hg. Immediately after cross-clamping the aorta, all piglets were cooled to a systemic temperature of 26°C, and warming to 37°C was begun 16 minutes before aortic unclamping. All piglets were weaned from CPB with no inotropic support 30 minutes after aortic unclamping. After arterial blood gases, Ca 2+, and K+ were normalized, and final functional and biochemical measurements were made 30 minutes later.


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  		Table 1. Warm Blood Cardioplegic Solution

 

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  		Table 2. Multidose Blood Cardioplegic Solution

 
Experimental groups
Warm versus cold induction
These investigations were done to determine the effects of cardioplegic induction temperature in normal (nonhypoxic) neonatal hearts, not subjected to hypoxia before cardioplegic ischemia, as well as in hypoxic hearts, to mimic a cyanotic ventricle undergoing surgical correction during an interval of aortic clamping. Both warm and cold cardioplegic induction solutions contained the amino acids Asp and Glut.

Nonhypoxic (normal) studies

  1. Cold Induction (group 1). In 5 piglets, the cardioplegic induction was given cold (4°C).
  2. Warm Induction (group 2). In 5 piglets, the cardioplegic induction was given warm (37°C).

Hypoxic (stressed) studies
Twenty-six other piglets underwent 60 minutes of ventilator hypoxia by lowering the fraction of inspired oxygen to 8% to 10%, producing an arterial partial pressure of oxygen of 25 to 35 mm Hg and an oxygen saturation of 65% to 70%. Before hypoxemia, piglets were transfused as necessary to increase their hematocrit to greater than 35%. This simulates the chronic adaptive change of erythrocytosis and increases oxygen carrying capacity, thereby allowing ischemia to be avoided during hypoxia [11, 16, 17]. At the end of 60 minutes, all piglets were placed on CPB (fraction of inspired oxygen, 100%) for 5 minutes to produce a reoxygenation injury, and then divided into three groups [14, 15, 17].

  1. Hypoxia–Reoxygenation Control (group 3). To evaluate the effect of reoxygenation of hypoxic (stressed) piglets, 6 underwent 70 minutes of CPB alone without subsequent cardioplegic arrest.
  2. Cold Induction (group 4). Five hypoxic (stressed) piglets underwent 70 minutes of cardioplegic arrest and received a cold (4°C) induction using the cardioplegia protocol outlined above.
  3. Warm Induction (group 5). Five hypoxic (stressed) piglets underwent 70 minutes of cardioplegic arrest and received a warm (37°C) induction using the cardioplegia protocol outlined above.

Amino acids and their mechanism of action
These investigations where performed to determine whether amino acid enrichment of the warm induction cardioplegic solution was necessary, as well as whether their mechanism of action was by means of metabolic enhancement (increased oxygen consumption [MO2], ATP) or vasodilation. Cardioplegic solution was delivered using the same protocol outlined above except that amino acids were not added to the warm induction solution (Table 1). These piglets (groups 6 and 7) were then compared with piglets receiving a warm induction with Asp and Glut (group 5 above). All piglets were subjected to a 60-minute hypoxic stress before cardioplegic arrest using the protocol outlined above.

  1. No Aspartate and Glutamate, No Nitroglycerin (group 6). In 5 hypoxic (stressed) piglets, the warm induction cardioplegic solution was not supplemented with Asp, Glut, or NTG (Table 1).
  2. No Aspartate and Glutamate, Nitroglycerin (group 7). In 5 hypoxic (stressed)piglets, warm induction cardioplegic solution was supplemented with the vasodilator NTG (Table 1), to determine whether amino acids enhance protection through vasodilation.

Myocardial performance
Left ventricular pressure and conductance catheter signals were amplified and digitized to inscribe left ventricular pressure-volume loops after first correcting for parallel conductance (myocardial tissue and blood viscosity) using hypertonic saline solution, as previously described [11, 16]. A series of pressure-volume loops was generated under varying conditions by transient occlusion of the inferior vena cava during an 8-second period of apnea. Measurements were made before hypoxia or bypass (baseline) and 30 minutes after CPB was discontinued. The end-systolic and end-diastolic pressure-volume relationship and the preload recruitable stroke work relationship were analyzed with the use of a computer graphics program, as previously described [11, 16]. Functional measurements are expressed as percent recovery of baseline values with each piglet acting as its own control. After final hemodynamic measurements, all piglets were placed back on CPB, and transmural left ventricular biopsies were obtained. Endocardial and epicardial portions were separated, frozen quickly in liquid nitrogen, and stored for biochemical analysis. A separate sample was obtained for myocardial water.

Physiologic measurements
Coronary vascular resistance
Coronary vascular resistance was determined during each cardioplegic infusion by measuring coronary sinus pressure and cardioplegic flow once a constant infusion rate with an aortic root pressure between 40 and 50 mm Hg was achieved. Coronary vascular resistance was calculated, as previously described, as the change in pressure across the coronary vascular bed, divided by the cardioplegic flow rate, multiplied by 80, and expressed as dynes per second per centimeter raised to the fifth power [11, 16].

Myocardial oxygen consumption
After cardioplegic arrest, blood was obtained at 1-minute intervals from the cardioplegic line and coronary sinus during the 5 minutes of cardioplegic induction, and MO2 was determined as previously described [7]. The cumulative 5-minute MO2 was determined by the sum of the individual 1-minute values and expressed per 100 g of heart tissue, which was determined by weighing the left ventricle at the conclusion of the experiment.

Biochemical analysis
Adenosine pool
Myocardial samples were crushed in a liquid nitrogen-cooled mortar and pestle and lyophilized. The adenosine pool was determined as described previously [11, 16]. Adenosine triphosphate levels are expressed as micrograms per gram of dry tissue.

Myeloperoxidase activity
Quantitative myeloperoxidase activity was determined as described previously [18]. Enzyme activity is expressed as the change in optical density units per minute per milligram of tissue protein.

Myocardial water
Ventricular samples were placed in preweighed vials and dried to a constant weight at a temperature of 85°C. The percent myocardial water was calculated using the following formula:

Statistics
Data were analyzed using JMP V2.0 (SAS Institute, Inc, Cary, NC) on a Macintosh IIVX computer (Apple Inc, Cupertino, CA). Paired Student’s t test and one-way analysis of variance was used for comparison of variables among experimental groups. If the analysis of variance revealed a significant interaction, pairwise tests of individual group means were compared by means of multiple comparisons (Tukey’s test) using a level of significance of p less than 0.05 and p less than 0.01. Group data are expressed as mean ± standard error of the mean.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
There was no difference between groups for before intervention (baseline) values (slopes) of left ventricular contractility (34 ± 2 mm Hg/mL), diastolic compliance (0.04 ± 0.01), or preload recruitable stroke work (72 ± 2 {int}[mm Hg · mL]/mL). All piglets remained stable during the 60 minutes of hypoxia.

Hemodynamic and physiologic measurements
Results are depicted in Figures 1–7. There was no change or difference in the x-axis intercept point (V0) for end-systolic elastance (before, 6.5 ± 0.1; after, 6.4 ± 0.1) or preload recruitable stroke work (before, 10.4 ± 0.1; after, 10.6 ± 0.2) before (baseline) and after CPB in any experimental group. Therefore, the change in slope of end-systolic elastance and preload recruitable stroke work can be interpreted to express variability in the contractile state of the myocardium compared with control (baseline) values.



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  		Fig 1. Warm versus cold cardioplegic induction: recovery of left ventricular systolic function in hypoxic hearts undergoing reoxygenation on cardiopulmonary bypass without ischemia or 70 minutes of cardioplegic arrest with an aspartate/glutamate (Asp/Glu) -enriched cardioplegic induction. Contractility is measured by the end-systolic elastance (EES) and expressed as a percentage of control (baseline) values. *p < 0.001.

 
Nonhypoxic (normal) hearts
There was complete preservation of myocardial function in all nonhypoxic (normal) hearts, with no difference (p > 0.2) between cold (group 1) or warm (group 2) cardioplegic induction regarding post-CPB systolic function (100% ± 2% versus 104% ± 3%), diastolic stiffness (162% ± 8% versus 156% ± 6%), or preload recruitable stroke work (100% ± 2% versus 102% ± 3%).

Hypoxia (stressed) hearts
Warm Versus Cold Induction. Hypoxia followed by reoxygenation without ischemia (stressed controls, group 3) produced an injury that decreased systolic contractility (51% ± 2%), increased diastolic stiffness (260% ± 15%), and reduced preload recruitable stroke work (54% ± 3%). Compared with hypoxia–reoxygenation controls (group 3), cold cardioplegic induction with amino acids (group 4) did not alter the hypoxia–reoxygenation injury, as post-CPB systolic function (51% ± 2% versus 51% ± 3%), diastolic stiffness (260% ± 15% versus 241% ± 5%), and preload recruitable stroke work (54% ± 3% versus 51% ± 2%) were not statistically different (p > 0.2). Conversely, compared with cold induction (group 4), warm cardioplegic induction with amino acids (group 5) repaired the injury caused by hypoxia and reoxygenation, resulting in improved post-CPB myocardial systolic (51% ± 3% versus 105% ± 3%;p < 0.001) and global myocardial function (51% ± 2% versus 103% ± 2%; p < 0.001) and reduced diastolic compliance (241% ± 13% versus 154% ± 7%; p < 0.001; Figs 1 and 2).



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  		Fig 2. Warm versus cold cardioplegic induction: postbypass left ventricular diastolic compliance in hypoxic hearts undergoing reoxygenation on cardiopulmonary bypass without ischemia or 70 minutes of cardioplegic arrest with an aspartate/glutamate (Asp/Glu) -enriched cardioplegic induction. Compliance is measured by the end-diastolic pressure-volume relationship and expressed as a percentage of stiffness compared with control (baseline) values. Note that there is a marked increase in diastolic stiffness in hypoxic hearts given a cold induction, whereas warm induction allowed for cellular repair of the hypoxia–reoxygenation injury, resulting in a diastolic compliance that is not statistically different from nonhypoxic (normal) hearts undergoing cardioplegic arrest. *p < 0.001.

 
Amino Acids and Their Mechanism of Action. Warm induction without amino acid enrichment (No Asp/Glut, No NTG, group 6; or No Asp/Glut, NTG, group 7) was unable to resuscitate the hypoxically injured heart, resulting in a similar (p > 0.2) depression of systolic contractility (end-systolic elastance, 51% ± 2% versus 51% ± 3%), preload recruitable stroke work (51% ± 3% versus 52% ± 2%), and increase in diastolic stiffness (243% ± 8% versus 238% ± 9%). These values were also comparable (p > 0.2) to hearts undergoing hypoxia and reoxygenation without ischemia (group 3) or cold induction with amino acids (group 4; Figs 3 and 4).



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  		Fig 3. Amino acids and their mechanism of action: recovery of left ventricular systolic function in hypoxic hearts receiving a warm cardioplegic induction (with or without aspartate/glutamate [Asp/Glu]) as measured by end-systolic elastance (EES) and expressed as percentage of control (baseline). Nitroglycerin (NTG) was added in one group of piglets not receiving aspartate/glutamate. *p < 0.001.

 


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  		Fig 4. Amino acids and their mechanism of action: postbypass left ventricular diastolic compliance in hypoxic hearts receiving a warm cardioplegic induction (with or without aspartate/glutamate [Asp/Glu]) as measured by the end-diastolic pressure-volume relationship and expressed as percentage of stiffness compared with control (baseline). Nitroglycerin (NTG) was added in one group of piglets not receiving aspartate/glutamate. *p < 0.001.

 
Myocardial oxygen consumption
Warm Versus Cold Induction. As would be expected in normal (nonhypoxic) hearts, MO2 (expressed as milliliters of oxygen per 100 grams per 5 minutes) during cardioplegic induction was essentially at basal metabolic rates for the arrested heart (group 1, cold, 4.6 ± 0.1 mL · 100 g-1 · 5 min-1; group 2, warm, 5.2 ± 0.2 mL · 100 g-1 · 5 min-1). The cumulative MO2 for the 5 minutes of induction was slightly lower in the cold induction group, but this is appropriate as the metabolic rate is decreased by hypothermia [8]. With cold induction, there was no increase (p = 0.2) in MO2 in hypoxic compared with nonhypoxic (normal) hearts (4.9 ± 0.2 mL · 100 g-1 · 5 min-1 hypoxic, group 4, versus 4.5 ± 0.1 mL · 100 g-1 · 5 min-1 nonhypoxic, group 1). Conversely, there was a slight increase in the MO2 during warm (amino acid-enriched) cardioplegic induction in hypoxic compared with nonhypoxic (normal) hearts (6.9 ± 0.3 mL · 100 g-1 · 5 min-1 hypoxic, group 5, versus 5.2 ± 0.2 mL · 100 g-1 · 5 min-1 nonhypoxic, group 2; p = 0.002).

Amino Acids and Their Mechanism of Action. Myocardial oxygen consumption was not different (p > 0.2) in hypoxic hearts given a warm cardioplegic induction (6.9 ± 0.3 mL · 100 g-1 · 5 min-1 Asp/Glut, group 5, versus 6.5 ± 0.3 mL · 100 g-1 · 5 min-1 No Asp/Glut, No NTG, group 6, versus 7.3 ± 0.4 mL · 100 g-1 · 5 min-1 No Asp/Glut, NTG, group 7), indicating that the metabolic activity during warm induction was the same with or without amino acid enrichment. Furthermore, the MO2 during warm induction in hypoxic hearts (groups 5 through 7) was only slightly higher than the normal basal metabolic requirements of the normothermic (37°C) heart [8].

Coronary vascular resistance
Warm Versus Cold Induction. Coronary vascular resistance was, as expected, increased by hypothermia. However, compared with nonhypoxic (normal) hearts, there was a significant increase in CVR during each cardioplegic infusion in hypoxic hearts given a cold cardioplegic induction (Fig 5). In contrast, there was no significant difference in CVR between nonhypoxic (group 2) and hypoxic (group 5) piglets receiving an amino acid-enriched warm induction (Fig 6).



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  		Fig 5. Warm versus cold cardioplegic induction: coronary vascular resistance (CVR) during each cardioplegic infusion in hypoxic and nonhypoxic hearts given an aspartate/glutamate-enriched cold induction. Note that there is a marked rise in coronary vascular resistance when cold induction is used in hypoxic hearts, implying a derangement in vascular function. *p < 0.001.

 


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  		Fig 6. Warm versus cold cardioplegic induction: coronary vascular resistance (CVR) during each cardioplegic infusion in hypoxic and nonhypoxic hearts given an aspartate/glutamate-enriched warm induction. Note that there is no difference in coronary vascular resistance in hearts given a warm induction, indicating preservation of vascular function despite a hypoxic stress. *p = not significant.

 
Amino Acids and Their Mechanism of Action. The CVR during warm induction was significantly increased if the warm induction solution did not contain amino acids or NTG (group 6). However, NTG was an effective vasodilator as the CVR was reduced with NTG enrichment (group 7; Fig 7).



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  		Fig 7. Amino acids and their mechanism of action: coronary vascular resistance (CVR) measured during cardioplegic induction in hypoxic piglets receiving a warm induction with or without aspartate/glutamate (Asp/Glu) -enrichment. Nitroglycerin (NTG) was added in one group of piglets not receiving aspartate/glutamate. *p < 0.001 versus all groups; **p < 0.001 versus no NTG.

 
Tissue studies
Results are summarized in Table 3. There was no change in ATP or the ATP to adenosine diphosphate ratio in nonhypoxic hearts, independent of the induction temperature. In hypoxic hearts, a substrate-enriched warm induction (group 5) preserved ATP at the same level as hearts subjected to hypoxia–reoxygenation without ischemia (group 3) or piglets not subjected to the stress of hypoxia (groups 1 and 2). Conversely, endocardial ATP levels were reduced in hypoxic hearts given a cold induction (group 4) or a warm cardioplegic induction without amino acids (groups 6 and 7). Furthermore, the ATP to adenosine diphosphate ratio, which reflects the ability of mitochondria to phosphorylate adenosine diphosphate to ATP, was markedly reduced in these same groups (groups 4, 6, and 7), suggesting mitochondrial damage. This may be why the ATP levels were depressed. These hearts also had the highest amount of myocardial water and myeloperoxidase activity, further supporting a greater cellular injury.


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  		Table 3. Endocardial Tissue and Myocardial Oxygen Uptake

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
This study demonstrates that in an intact animal model simulating the operating room, (1) excellent myocardial protection is achieved in nonhypoxic (normal) neonatal hearts using either warm or cold cardioplegic induction, (2) in hypoxic hearts, cold cardioplegic induction preserves myocardial function at preischemic levels, but is unable to modify the hypoxia–reoxygenation (stress) injury, and (3) warm cardioplegic induction facilitates myocardial repair of the hypoxia–reoxygenation stress injury, resulting in complete return of myocardial function. However, warm induction cardioplegia is only effective in resuscitating the hypoxic neonatal heart when it is enriched with the amino acids Asp and Glut, and, unlike adult hearts, the mechanism of action of amino acid supplementation during warm induction in neonates remains unknown, but does not appear to be secondary to increased metabolic activity or vasodilation.

A brief (5 minutes) infusion of warm blood cardioplegic solution can be used as a form of active resuscitation in energy-depleted (ischemic) adult hearts that must undergo subsequent aortic clamping [7, 8]. Normothermia (37°C) optimizes the rate of cellular repair, and enrichment with the amino acids Asp and Glut improves oxygen utilization capacity, resulting in improvement in postoperative functional recovery and patient survival [8, 19]. This extra oxygen is used to repair ischemic cell damage, as well as to replete energy stores, thereby allowing the myocardium to better tolerate the obligatory period of aortic cross-clamping needed for cardiac repair. Warm blood cardioplegic induction is therefore often a part of the myocardial protection strategy in adult hearts subjected to a preoperative ischemic stress or hemodynamic instability [7, 8]. Because preoperative ischemia is rare in the neonatal heart, warm induction is seldom used in pediatric heart operations, for which its role remains largely undefined.

In the nonhypoxic (normal) heart, cardioplegic induction temperature did not alter protection, as there was complete preservation of myocardial function and metabolic activity with either warm or cold induction. There was also no significant increase in MO2 over basal metabolic rates during cardioplegic induction. This is not surprising as nonhypoxic (normal) hearts should not need to be resuscitated, and cold blood cardioplegia has been shown to provide excellent myocardial protection in normal neonatal hearts undergoing 2 hours of aortic cross-clamping [1, 2, 20]. However, in clinical practice, most pediatric hearts are stressed as a result of hypoxia or a pressure-volume overload, and therefore normal hearts are probably uncommon, especially in the neonatal population.

Hypoxia followed by reoxygenation without aortic clamping (stress controls, group 3) produced an oxygen-mediated injury that depressed systolic and global myocardial function to approximately 50% of pre-CPB levels and increased diastolic stiffness significantly [11, 14, 15, 17]. Cold cardioplegic induction prevented further damage, although it did not alter the hypoxia–reoxygenation injury, as post-CPB function was identical to that of hypoxia–reoxygenation without ischemia. Conversely, providing a warm induction facilitated cellular repair of the hypoxic–reoxygenation injury, resulting in complete preservation of myocardial function. However, the benefits of warm induction were only realized if the cardioplegic solution contained the amino acids Asp and Glut; furthermore, the MO2 was not significantly increased over basal metabolic rates or over cardioplegic solution without amino acids [8]. Therefore, unlike ischemic (stressed) adult hearts, which increase MO2 fivefold during warm induction, the primary mechanism of amino acid supplementation in hypoxic (stressed) neonates does not appear to be secondary to increased metabolic activity [8]. This may be related to the fact that ischemia and hypoxia result in different myocardial injuries [14]. With ischemia, there is significant depletion of ATP, resulting in the loss of cellular ionic gradients [8]. Warm induction allows the heart to generate substantial quantities of ATP, making it possible to reestablish these ionic gradients, which explains the large increase in MO2 seen in ischemic adult hearts during warm induction [7, 8]. Conversely, in our acute hypoxic model there is no ischemia, as oxygen delivery is maintained during hypoxia, preserving ATP levels [11, 17]. Because ATP levels are not reduced, there should be no loss of cellular ionic gradients. Therefore, MO2 during warm induction does not need to be significantly increased over basal levels, because cellular ionic gradients do not need to be reestablished. This explains why the ATP levels were the same in hearts given a warm amino acid-enriched cardioplegic induction as they were in hypoxia–reoxygenation piglets not subjected to ischemia. Energy levels were preserved, but not enhanced with amino acid enrichment. In contrast, the lower post-CPB ATP to adenosine diphosphate ratio in hypoxic piglets receiving cold induction (group 4) or warm induction without amino acids (groups 6 and 7) suggests that the cell has sustained a mitochondrial injury, resulting in the inability to maintain ATP levels during ischemia.

Several studies have advocated using a warm induction in pediatric patients to prevent a rapid cooling contracture [1, 21, 22]. This injury can also be prevented by cooling slowly or using a hypocalcemic cardioplegic solution, both of which were used in this study. It is possible that hypoxically stressed hearts may be more susceptible to cold contracture. However, it is unlikely that this mechanism explains our findings, as warm induction without amino acids resulted in the same function as cold induction or hypoxia–reoxygenation without ischemia. The data therefore imply that warm induction with amino acids repairs the cellular injury caused by hypoxia followed by reoxygenation, whereas cold induction, or warm induction without amino acids, prevents further damage during aortic clamping but does not alter the injury.

Coronary vascular resistance was measured during cardioplegic infusions to determine the effect of induction temperature on vascular function. Although CVR increases with hypothermia secondary to reduced metabolic demands, it should not be affected by hypoxia, as our experimental model of acute hypoxia does not cause ischemia, and hearts are reoxygenated before cardioplegic arrest. As expected, the CVR was similar in hypoxic and nonhypoxic hearts given a substrate-enriched warm cardioplegic induction. In contrast, there was a significant and sustained increase in CVR in hypoxic hearts given a cold induction. This increase in CVR can be caused by either vascular dysfunction or myocardial edema. However, it appears to reflect a derangement in vascular function, as there was no significant difference in myocardial edema between hypoxic hearts given a substrate-enriched warm or cold induction (groups 4 and 5). A drawback of this study is that vascular function was not independently assessed after cardioplegic arrest. An elevated CVR is therefore suggestive of a vascular injury, but this cannot be proven because specific tests of endothelial cell function were not performed.

Amino acids may enhance cardioplegic delivery through vasodilation [8, 9]. To determine whether this mechanism was responsible for their beneficial effect, the vasodilator NTG was added to the warm induction cardioplegic solution in one group of piglets (group 7). Nitroglycerin was an effective vasodilator as the CVR during warm induction was lowest in this group of animals. However, myocardial functional recovery was not improved with NTG, whereas there was complete recovery with amino acid enrichment. This implies that vasodilation is not the primary mechanism responsible for the functional improvement seen with amino acids. Rather, it suggests that amino acids reduce CVR by preventing a vascular injury and maintaining normal vascular function, whereas NTG causes vasodilation but does not prevent myocardial injury.

We can speculate as to the mechanism of action of amino acid enrichment in hypoxic neonatal hearts by examining the myeloperoxidase activity, which is a quantitative measurement of white blood cell sequestration. Hypoxia activates vascular endothelial cells, resulting in increased white blood cell adherence [23, 24]. Once bound, white blood cells release oxygen free radicals and proteases, which damage the vascular as well as the myocardial cell. By increasing nitric oxide production, certain substances, such as L-arginine, can inhibit white blood cell adherence, leading to improved myocardial function and reduced myeloperoxidase activity [18, 24, 25]. Although Glut is not known to stimulate nitric oxide in the heart, it does increase nitric oxide levels in the brain [26]. Perhaps Glut or Asp interacts with an unknown receptor, increasing nitric oxide release in myocardial tissue. Alternatively, Asp and Glut are known to have antioxidant properties, and have been shown to reduce oxygen free radical damage in hypoxic hearts [27]. Avoidance of an oxidant injury would also reduce the tissue myeloperoxidase activity, as well as preserve vascular and myocardial function. Evidence that amino acids can act through a mechanism other than by increasing energy production could explain why some investigators have shown no active incorporation of amino acids into the Krebs cycle, despite substantial evidence that they improve myocardial protection [1, 8, 28]. Although the mechanism of action therefore remains uncertain, this report provides direct evidence that the ability of amino acid enrichment to improve cellular function is more than just the ability to replenish depleted energy stores.

Another unanswered question is whether both amino acids are needed. We used both because they have been shown to be synergistic in adults [8]. Several surgeons have, however, expressed concern that because Glut is a neurotransmitter, it may be detrimental, especially during circulatory arrest [9, 29]. This is unlikely as (1) Glut levels in the brain are approximately 100 times those of the serum, (2) enriching cardioplegic solutions with Glut only raises serum levels 2 to 3 times, which is insignificant compared with neural tissue, and (3) Glut does not cross the intact blood–brain barrier [29]. In addition, this and other studies suggest that amino acids are effective only when given warm [8]. This further decreases any potential risks, as amino acids are not administered during hypothermic circulatory arrest,.

Our model of acute hypoxia does not allow for the chronic adaptive changes that may occur in cyanotic newborns, although it does subject the heart to a clinically relevant stress. Using stressed hearts is important, even if the model does not exactly simulate the clinical setting, because stressed hearts are less tolerant of ischemia, and more sensitive to changes in the method of cardioplegic protection. They therefore provide information concerning the patients most vulnerable to postoperative dysfunction. This is why adult studies often use an acute ischemic stress to investigate cardioplegia strategies, even though it does not exactly mimic chronic angina or cardiogenic shock [8]. Several studies have documented a similar oxygen-mediated injury with reoxygenation of the chronically hypoxic infant, even in the absence of ischemia, and we recently documented an identical injury in 21 cyanotic infants using the same biochemical tests as our acute experimental studies [11, 15, 17, 23, 30]. Furthermore, our experimental model of acute hypoxia does not cause ischemia or depletion of ATP stores, whereas chronically cyanotic hearts may be predisposed to ischemia with accelerated depletion of high-energy compounds during periods of increased myocardial oxygen demands [11, 12, 17, 31, 32]. It is therefore possible that warm induction is even more important in cyanotic infants, inasmuch as in the presence of ATP depletion (ischemia), warm induction with amino acid enrichment has been shown to improve metabolic and functional recovery by repaying the energy debt [8].

In summary, this study demonstrates that both warm and cold cardioplegic induction provide good myocardial protection in nonhypoxic (normal) neonatal hearts. Conversely, when the heart is subjected to a hypoxic stress, the method of cardioplegic induction takes on increased importance, with warm substrate-enriched cardioplegic induction providing superior myocardial protection. The mechanism by which these amino acids work, however, remains uncertain, but is not secondary to increased metabolic activity or vasodilation. Inasmuch as cyanotic children often have depressed myocardial function after apparently successful surgical repair, this study suggest that surgeons should consider using a substrate-enriched warm cardioplegic induction to optimize myocardial protection [1, 2, 5, 6].


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
Michael T. Kronon, MD, was supported in part by the Pillsbury Fellowship.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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Accepted for publication April 1, 2000.




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