Ann Thorac Surg 1995;59:1435-1440
© 1995 The Society of Thoracic Surgeons
Pharmacologic Support With High-Energy Phosphate Preservation in the Postischemic Neonatal Heart
Ara K. Pridjian, MD,
Edward D. Frohlich, MD,
Clifford H. VanMeter, MD,
P. Michael McFadden, MD,
John L. Ochsner, MD
Section of Cardiovascular Surgery, Ochsner Clinic, New Orleans, Louisiana
Accepted for publication January 21, 1995.
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Abstract
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Milrinone improves function in failing adult hearts. This study examined its effect on immature myocardium. Using an isolated working neonatal rabbit heart preparation, we measured myocardial function, high-energy compounds, and cyclic adenosine monophosphate. Hearts were subjected to 1 hour of normothermic ischemia, 10 minutes of reperfusion with Ringer's solution, and 30 minutes of reperfusion with either unaltered Ringer's, Ringer's with dobutamine (0.1 µg/mL), or Ringer's with milrinone (1 µg/mL). These hearts were compared with each other, with a control group continuously perfused for 70 minutes, and with a group of hearts that were made ischemic and reperfused for only 10 minutes. There was a progressive decline in adenosine triphosphate levels measured in hearts from the groups receiving 10 and 40 minutes of reperfusion with unaltered perfusate, and cardiac output fell to 82% ± 4% of preischemic control in the latter group. When either dobutamine or milrinone was added to the reperfusion solution, postischemic myocardial function was restored completely, and the loss of adenosine triphosphate with reperfusion was halted. Cyclic adenosine monophosphate level was highest in ischemic/40-minute reperfused hearts, and there was no measurable increase in cyclic adenosine monophosphate level in the group of hearts receiving milrinone. The mechanism of preservation of high-energy stores with inotropic agents is not known but may involve potentiation of mitochondrial oxidative phosphorylation. Beginning pressor support early in the reperfusion period may help preserve adenosine triphosphate, and use of milrinone may be an important new adjunct in the treatment of neonates with myocardial dysfunction after congenital cardiac operations.
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Introduction
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See also page 1439.
Palliation or repair of complex congenital cardiac anomalies is now performed routinely in the neonatal period in experienced centers. Although results have been excellent, patients usually need inotropic support during the time that myocardial edema resolves, adenosine triphosphate (ATP) level is restored, pulmonary vascular resistance falls, and renal function recovers. To this end, infusions of one or a combination of catecholamine inotropes, including dopamine, dobutamine, or epinephrine, often are used in the perioperative period. Although these agents improve systolic function, they increase myocardial oxygen consumption. Also, at high doses 
-adrenergic effects predominate, resulting in decreased peripheral perfusion and reductions in blood flow to pulmonary, renal, and mesenteric beds. Neonates often have little reserve, and even subtle alterations in blood flow can contribute to hypoxemia, organ failure, and acidosis.
Amrinone and milrinone are phosphodiesterase inhibitors that do not increase myocardial oxygen consumption [1, 2]. Amrinone lowers systemic and pulmonary vascular resistance and improves diastolic myocardial function after congenital cardiac operations. However, compared with catecholamine agents, amrinone produces little direct improvement in systolic function, and amrinone may cause thrombocytopenia when used for an extended period of time [2–4]. In addition, it is often necessary to use - or ß-adrenergic agents in conjunction with amrinone to bring cardiac output and systolic blood pressure into acceptable ranges [5]. Milrinone is an analogue of amrinone that significantly improves both systolic and diastolic function, does not produce thrombocytopenia, and also decreases pulmonary vascular resistance [6]. For these reasons milrinone might be an ideal pressor agent for some neonates. We designed this experiment to assess hemodynamic and biochemical effects of milrinone in the postischemic neonatal heart, and to compare these with the effects of dobutamine, a commonly used agent.
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Material and Methods
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Preparation
New Zealand White rabbits aged 7 to 10 days were used in an isolated working heart preparation. After induction of anesthesia using ketamine (100 mg/kg) and xylazine (10 mg/kg), animals were heparinized (150 IU/kg) and hearts were excised and placed in a 4°C perfusate bath. Aortas were cannulated and hearts were mounted for perfusion in classic retrograde Langendorff mode. Modified Krebs-Ringer bicarbonate solution [7] warmed to 37°C and bubbled with 95% O2/5% CO2 gas (pH 7.44 to 7.48; oxygen tension, 450 to 550 mm Hg; 300 to 310 mOsm/L) was delivered at 50 mm Hg pressure. The perfusate contained (in millimoles/liter) the following: NaCl, 118; NaHCO3, 25; KH2PO4, 0.66; KCl, 4; MgCl2, 1.2; CaCl2, 2.4; and dextrose, 11.1. Left atria were cannulated, and after 10 minutes of perfusion, hearts were converted to working mode. Perfusate entered the left atrium, traversed the mitral valve into the left ventricle, then was ejected through the aortic valve into an aortic outflow column with an elasticity chamber. Preload was adjusted to 10 and 15 mm Hg by changing the height of the left atrial inflow column, and afterload was kept at 50 mm Hg. Aortic and coronary flows were measured directly by timed volumetric measurements. Cardiac output was calculated as the sum of aortic and coronary flows, and coronary vascular resistance (mm Hg • min/mL) was determined by dividing the aortic pressure (50 mm Hg) by the coronary flow. The percent change in resistance after ischemia and reperfusion was determined for each heart. Hearts were maintained at 37°C in a sealed, water-jacketed chamber.
Protocol
Hearts were allowed 20 minutes to equilibrate in working mode, and preischemic aortic and coronary flows were measured at left atrial pressures of 10 and 15 mm Hg. The left atrial and aortic lines then were clamped, and hearts underwent 1 hour of ischemia at 37°C. Hearts then were reperfused for 5 minutes in retrograde nonworking mode and 5 minutes in working mode with unaltered perfusate, followed by 30 minutes in working mode with either perfusate, perfusate with dobutamine (0.1 µg/mL), or perfusate solution with milrinone (1 µg/mL). Preliminary dose-response curves demonstrated that these were the lowest concentrations necessary to return cardiac output to preischemic levels after 1 hour of ischemia. Functional measurements were made again, and percent recoveries of preischemic aortic flow and cardiac output were determined. To discover if there was any loss of function inherent in the isolated heart model, a separate group was perfused continuously, and function was determined at 30 and 70 minutes. Another group of hearts was made ischemic and reperfused for a total of 10 minutes (5 minutes nonworking and 5 minutes working) with unaltered perfusate to determine the contents of high-energy phosphates just before inotrope infusions were begun. Diagramatic comparison of groups is given in Figure 1
. There were 11 control hearts, 10 ischemic/40-minute reperfused hearts, 13 ischemic/10-minute reperfused hearts, 9 dobutamine hearts, and 15 milrinone hearts.
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Fig 1. . Experimental groups: A separate group was perfused continuously for 70 minutes. (RP = reperfusion.)
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After perfusion, hearts were frozen in liquid nitrogen for later assay of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) using high-pressure liquid chromatography. Cyclic AMP (cAMP) was measured using a commercially available enzyme-linked immunosorbent assay (single range cyclic AMP enzyme immunoassay kit #8-6310; PerSeptive Diagnostics, Inc). Results are expressed as micromoles per gram of dry tissue ± standard error of the mean for ATP, ADP, and AMP, and picomoles per milligram of dry tissue ± standard error of the mean for cAMP.
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 85-23, revised 1985).
Statistics
Statistical analysis of biochemical and functional data was performed using analysis of variance to compare group means. Aortic flow, cardiac output, and coronary flow of control, ischemic/40-minute reperfused, dobutamine, and milrinone groups were compared. For biochemical data, ATP, ADP, AMP, and cAMP levels of all groups including ischemic/10-minute reperfused hearts were used in the analysis of variance. Multiple applications of the Tukey-Kramer test were made to determine the statistical significance of differences between groups. Raw data and percent recoveries for preischemic and postischemic function are given. A p value less than 0.05 was considered significant.
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Results
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Table 1 shows the percent recoveries, and Appendix 1 gives the raw data for aortic flow and cardiac output at left atrial pressures of 10 and 15 mm Hg. All data are presented as mean ± standard error of the mean. Aortic flow and cardiac output fell to approximately 80% of preischemic levels after ischemia and 40 minutes of reperfusion with unaltered Ringer's solution. Hearts receiving milrinone recovered 95% ± 4% of preischemic cardiac output at both 10 and 15 mm Hg preloads. Similarly, hearts in the dobutamine group recovered 103% ± 7% of preischemic cardiac output at 10 mm Hg and 108% ± 7% at 15 mm Hg.
Preischemic and postischemic coronary flow, coronary vascular resistance, and the percent change in resistance are shown in Table 2. Resistance decreased 8% ± 1% between 30 and 70 minutes in control hearts, increased 13% ± 6% in ischemic/40-minute reperfused hearts, and increased 22% ± 8% and 20% ± 5% in dobutamine and milrinone hearts, respectively.
Biochemical data are shown in Table 3. The level of ATP in hearts that were perfused continuously for 70 minutes measured 25.9 ± 1.6 µmol/g. After ischemia and 10 minutes of reperfusion, ATP level was 20.8 ± 1.7 µmol/g, and with 40 minutes of unaltered reperfusion ATP level fell to 9.1 ± 0.8 µmol/g. In contrast, at the end of reperfusion with dobutamine and milrinone, myocardial ATP levels were 22.4 ± 1.6 µmol/g and 22.2 ± 3.1 µmol/g, respectively. There was no difference between these and levels measured in nonischemic control hearts (25.9 ± 1.6 µmol/g). Cyclic AMP in milrinone (3.6 ± 0.5 pmol/mg), dobutamine (2.7 ± .2 pmol/mg), and ischemic/10-minute reperfused hearts (2.7 ± 0.2 pmol/mg) were all significantly lower than nonischemic controls (4.2 ± 0.5 pmol/mg). There was a small but statistically significant increase in cAMP level measured in hearts receiving milrinone (3.6 ± 0.5 pmol/mg) versus dobutamine (2.7 ± 0.2 pmol/mg). Elevation of cAMP above control levels was found only in the ischemic hearts receiving 40 minutes of unaltered reperfusion (5.7 ± 0.6 pmol/mg).
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Comment
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The finding that ATP was restored to control levels with either inotrope is both exciting and provocative, but unexpected. Catecholamine inotropes such as dobutamine are said to have ``oxygen-wasting'' effects [8]. One might also predict that by forcing increases in heart rate, contractility, and wall stress, catecholamines might ``waste'' ATP. Dobutamine increases intracellular calcium level, which strengthens the force of myocardial contraction, but also creates the burden of removing additional calcium from the cytosol to effect relaxation [9]. This is an energy-dependent process that requires ATP hydrolysis by cytosolic membrane and sarcoplasmic reticular calcium ATPases [10, 11].
Phosphodiesterase inhibitors improve contractility without a concomitant increase in myocardial oxygen consumption [2], so preservation of high-energy stores with milrinone is not especially surprising. The mechanism for the improvement in myocardial efficiency is not known definitively, but may involve an increase in the rate of sodium/calcium exchange, which does not require hydrolysis of ATP [12, 13]. Sodium/calcium exchange removes calcium from the cytoplasm for diastole, and this may be responsible for the lucitropic effect of the bipyridine compounds. One can reason, however, that neither phosphodiesterase inhibition nor sodium/calcium exchange is important in the preservation of high-energy stores, as high-energy compounds are preserved with infusion of either inotrope.
Both milrinone and dobutamine do increase intracellular calcium, and this is thought to be the basis of their salutary effects on systolic function. However, any effects of inotropic agents on calcium are superimposed on the postischemic pathologic calcium influx, which results in activation of autolytic enzymes, precipitation of proteins, and a decrease in mitochondrial oxidative phosphorylation [14–16]. It seems contradictory that using agents that actually increase intracellular calcium could improve metabolic and functional parameters in the postischemic heart. Independent of the effects on calcium mediated by adrenergic receptors or cAMP stimulation, we speculate that dobutamine and milrinone also may potentiate mitochondrial respiration with greater phosphorylation of ADP to ATP. Otherwise, simply raising intracellular calcium in the postischemic heart would not be expected to improve function and high-energy stores. Although it is also possible that either agent may inhibit 5`-nucleotidase, activity of this enzyme is low in the neonatal heart, and inhibition probably would not contribute much to the high-energy phosphate supply [17, 18].
Although cAMP is increased by some inotropic agents, these data suggest that elevation of cAMP is neither necessary nor sufficient to effect an improvement in ventricular function or to preserve high-energy phosphates. That there is no simple relationship between cAMP and myocardial contractile force has been shown by Frangakis and colleagues [19]. Using isolated perfused hearts and ventricular trabeculae from dog, guinea pig, and rat, they discovered four instances in which milrinone's effects on cAMP and contractile force did not correlate. In our study, in hearts receiving dobutamine, cAMP levels were low, whereas function and ATP were restored to control levels. Contrariwise, cAMP stores were actually greatest after ischemia and 40 minutes of unmodified reperfusion, whereas ventricular function was depressed and ATP stores were depleted. These findings are in agreement with others showing an increase in tissue levels of cAMP after ischemia [20, 21]. Also, there is evidence for different cellular compartments for cAMP [22]. The existence of compartments might explain the lack of relationship between total cellular cAMP and myocardial function. Admittedly, however, the existence of compartments enables interpretation of any data to the preference of the author. Immunocytochemical methods could be used to determine whether a compartmental theory should be embraced [23].
Theoretically, infusion of inotropic agents early in the reperfusion interval stresses a heart at a vulnerable time, when aerobic metabolism may not have resumed, and when concentration and electrochemical gradients may not have been reestablished. However, postischemic ion influx, intracellular and interstitial edema, and the pathologic changes that follow are progressive, becoming more severe as the reperfusion interval is extended. In the current study, administration of inotropes was begun relatively early in reperfusion. It is possible that this timing is important, so that the salutary effects of dobutamine and milrinone depend on beginning infusions before there is irreparable injury.
We noted an increase in coronary resistance with both inotropic agents. However, it is difficult to interpret this finding as there is near-maximal coronary vasodilation in the crystalloid-perfused heart model, and clinically, neither agent is known to increase coronary vascular resistance [24]. Nonetheless, this finding does suggest that in this study the functional and biochemical improvements seen with either agent are not simply due to an increase in coronary flow with improvements in oxygen and substrate delivery. A blood-perfused model might be better to assess the effects of inotropic agents on coronary vascular resistance in the postischemic myocardium, and hemodynamic profiles may have been slightly different with blood perfusion [25]. Nonetheless, we believed that using a crystalloid-perfused model was crucial for this study, as it allows tight control of variables such as the identity and concentration of metabolic substrates and the ionized calcium concentration.
There is often some degree of myocardial dysfunction after complex congenital cardiac procedures. An inotropic agent is chosen while considering the desired effects on myocardial systolic and diastolic function, as well as pulmonary and systemic vascular resistances. In addition to these observed effects, some inotropic agents also may have salutary effects on postischemic mitochondrial dysfunction and high-energy phosphate stores. Milrinone improves diastolic function, decreases pulmonary vascular resistance, and does not increase oxygen consumption above baseline levels. Under conditions when oxygen supply is limited, as is frequently the case in neonates with complex congenital cardiac defects, milrinone may be a very useful agent.
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Appendix 1.
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Table . Preischemic and Postischemic Aortic Flow and Cardiac Output (mL/min) With 30- and 70-Minute Control Heart Dataa
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Footnotes
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Address reprint requests to Dr Pridjian, Ochsner Clinic, 1514 Jefferson Highway, New Orleans, LA 70121.
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References
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Abstract
Introduction
