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J Thorac Cardiovasc Surg 1994;107:520-0526
© 1994 Mosby, Inc.

Cardiopulmonary Bypass, Myocardial Management, and Support Techniques

Developmental differences in myocardial protection in response to 5'-nucleotidase inhibition

Ann Arbor, Mich.

From the Thoracic Surgery Research Laboratory, Section of Thoracic Surgery, Department of Surgery, The University of Michigan School of Medicine, Ann Arbor, Mich.

Received for publication Feb. 2, 1993. Accepted for publication June 22, 1993. Address for reprints: Edward L. Bove, MD, 2120 Taubman Center, Box 0344, 1500 E. Medical Center Dr., Ann Arbor, MI 48109.

Abstract

Age-related differences in the activity of 5'-nucleotidase, an enzyme responsible for conversion of high-energy phosphates to their the diffusible precursors, may help to explain age-related differences in tolerance of global myocardial ischemia. Postischemic function and high-energy phosphate content were measured in the hearts of rabbits 7 to 10 days old (neonate), 30 to 40 days old (1 month), and 6 to 12 months old (adult). Hearts in each age group were subjected to 60 minutes of ischemia at 34° C either with no cardioplegia, with unmodified St. Thomas' Hospital cardioplegic solution, or with St. Thomas' Hospital cardioplegic solution with pentoxifylline, a 5'-nucleotidase inhibitor. These groups were compared with one another and with control hearts that were continuously perfused for 1 hour. In adults, addition of pentoxifylline to St. Thomas' Hospital cardioplegic solution restored adenosine triphosphate and total nondiffusible nucleotide levels to control values and improved recovery of cardiac output and developed pressure compared with results with unmodified St. Thomas' Hospital cardioplegic solution. In contrast, biochemical and functional parameters in neonatal hearts were not affected by either unmodified St. Thomas' Hospital cardioplegic solution cardioplegia or St. Thomas' Hospital cardioplegic solution with pentoxifylline. Functional recovery in neonatal hearts subjected to unprotected ischemia was superior to that in the older age groups. In 1-month-old hearts, St. Thomas' Hospital cardioplegia improved recovery compared with recovery after unprotected ischemia, but no incremental improvement in function or high-energy stores was seen with addition of pentoxifylline. The lack of effect of pentoxifylline on neonatal hearts suggests that there is a relative deficiency of 5'-nucleotidase in this age group. This may contribute to the improved functional recovery observed in unprotected hearts. Furthermore, addition of pentoxifylline to adult hearts appears to confer the benefits of low 5'-nucleotidase activity occurring naturally in the neonate. (J THORAC CARDIOVASC SURG 1994;107:520-6)

The immature heart tolerates ischemia and reperfusion better than the adult heart. Though this finding has been extensively reported in a variety of species, the biochemical basis has not been completely elucidated. ^1-4 In the adult heart, the enzyme 5'-nucleotidase (5'NT) catalyzes the degradation of adenosine monophosphate (AMP) and inosine monophosphate (IMP) nucleotides to their precursors, adenosine and inosine nucleosides. ^5-8 These diffusible forms are lost during reperfusion and are not available for repletion of energy stores. It has been postulated that because there is lower activity of 5'NT in the immature heart, there is less breakdown of high-energy phosphates than in adult hearts. ^{1, 2, 9} Although postischemic high-energy nucleotide loss may be blunted in the immature heart, there is nonetheless significant high-energy phosphate depletion and functional impairment after ischemia and reperfusion. ^{1, 2, 10, 11} Breakdown of AMP and IMP to adenosine and inosine by 5'NT has been shown to be the predominant mechanism of high-energy phosphate loss in the adult heart. ¹² In the immature heart, however, the relative contribution of 5'NT activity versus other mechanisms that may affect high-energy phosphate depletion is not known. The purpose of this study was to determine the consequences of 5'NT inhibition in the setting of global ischemia in neonatal, immature, and adult hearts.

METHODS

Experimental preparation
Hearts from newborn (7 to 10 days old), 1 month (30 to 40 days old), and adult (6 to 12 months old) New Zealand White rabbits were used. Hearts were perfused in working mode as described by Neely and associates. ¹³ After induction of anesthesia with 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 retrograde (Langendorff) perfusion. An oxygenated modified Ringer's solution ¹⁴ warmed to 37° C was delivered at 40, 50, and 80 mm Hg to newborn, 1 month, and adult hearts, respectively. A 3 µm microfilter was used in the perfusion circuit. The left atrium was cannulated, and after 10 minutes hearts were converted to working mode with perfusate entering the left atrium. The perfusate then flowed into the left ventricle and was ejected into an aortic outflow column against 40, 50, or 80 mm Hg in the three respective age groups. Aortic and coronary flows (milliliters per minute) were collected in graduated cylinders. A 3F high-fidelity catheter-mounted pressure transducer (model SPR-249, Millar Instruments, Houston, Tex.) introduced through the left atrial catheter was advanced into the left ventricle for measurements of left ventricular systolic and end-diastolic pressure. Developed pressure was calculated as the difference between systolic pressure and end-diastolic pressure.

Experimental protocol
After a 30-minute period of equilibration in the working mode, preischemic measurements were obtained. Heart rate, developed pressure, coronary flow, and aortic flow were measured. Cardiac output was calculated as the sum of aortic and coronary flows. A control group was subjected to unprotected ischemia at 34° C for 60 minutes. In the experimental groups, hearts were allowed a 30-minute equilibration period in the working mode followed by 1 hour of ischemia produced by clamping the left atrial line as the temperature of the water-jacketed chamber was lowered to 34° C. Coincident with the onset of global ischemia one group received unmodified St. Thomas' Hospital cardioplegic solution (in millimoles per liter: 110 NaCl, 10 NaHCO₃, 16 KCl, 16 MgCl₂, and 1.2 CaCl₂). The other group was given St. Thomas' Hospital cardioplegic solution with pentoxifylline (500 mg/L, Trental, Hoescht-Roussel, Somerville, N.J.).

Timed 3-minute infusions of cardioplegic solution were delivered at 40, 50, or 80 mm Hg pressure to the neonate, 1-month, and adult age groups. A second infusion of cardioplegic solution was delivered after 30 minutes. Hearts were reperfused at the end of the 60-minute ischemic interval for 10 minutes in the retrograde nonworking mode followed by 30 minutes in the working mode. Cardiac output and developed pressure were again measured and percent recovery of preischemic control was calculated. Hearts were then frozen in liquid nitrogen for later assay of myocardial contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), AMP, and IMP by high-performance liquid chromatography. A separate group of hearts, continuously perfused for 1 hour, was used to measure baseline high-energy phosphate concentrations for each age group. Results are expressed as nanomoles per milligram dry tissue plus or minus the standard error of the mean. The total nondiffusible nucleotides (TNN) were determined as the sum of ATP, ADP, AMP, and IMP.

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. 86-23, revised 1985).

Statistics
Statistical analysis was done by Student's t test and analysis of variance. Tukey-Kramer test was used to determine statistically significant differences between groups. A p value of less than 0.05 was considered significant. Raw data for preischemic and postischemic cardiac output and developed pressure are reported and used to make comparisons between groups.

RESULTS

The hemodynamic data for newborn, 1 month, and adult hearts are shown in Figs. 1 through 3 and in the appendix. In neonates (Fig. 1), recovery of cardiac output and developed pressure declined to 70% and 74% of their respective control values after unprotected ischemia, and hemodynamic performance was not improved by either the St. Thomas' Hospital or the modified St. Thomas' Hospital cardioplegic solution. In contrast, after 1 hour of unprotected ischemia, 1-month-old hearts (Fig. 2) were severely depressed, whereas hearts receiving St. Thomas' Hospital cardioplegic solution recovered 66% and 86% of cardiac output and developed pressure, respectively. The addition of pentoxifylline resulted in a worse recovery of cardiac output (39%) and developed pressure (75%) when compared with values in control hearts. Adult hearts were also severely damaged by unprotected ischemia (Fig. 3), but recovered 55% of cardiac output and 91% of developed pressure with unmodified St. Thomas' Hospital solution. When pentoxifylline was added, cardiac output improved to 78% and developed pressure was 99% of baseline value.

View larger version (34K): [in this window] [in a new window]   Fig. 1. Cardiac output and developed pressure before and after ischemia, St.Thomas' Hospital cardioplegia (CP), and St. Thomas' Hospital cardioplegia with pentoxifylline (PTX) in neonatal hearts.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Cardiac output and developed pressure before and after ischemia, St. Thomas' Hospital cardioplegia (CP), and St. Thomas' Hospital cardioplegia with pentoxifylline (PTX) in adult hearts.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Cardiac output and developed pressure before and after ischemia, St.Thomas' Hospital cardioplegia (CP), and St. Thomas' Hospital cardioplegia with pentoxifylline (PTX) in 1-month-old hearts.

DISCUSSION

Immature hearts tolerate ischemia and reperfusion better than those of adults, and myocardial protection for the neonate undergoing correction of congenital heart defects is seldom a problem. The mechanism of this apparently better tolerance of global ischemia is unknown, but may be attributable to differences in high-energy phosphate metabolism. In hearts that are injured by global ischemia, high-energy phosphates are broken down and may not be rapidly resynthesized after reperfusion. ¹⁰ If this loss of high-energy compounds occurs to a lesser degree in neonatal hearts than in adults, understanding the biochemical basis for this process may be helpful in improving myocardial protection among all age groups.

In the ischemic myocardium ATP is broken down to ADP, AMP, and IMP. These precursors are nondiffusible and can be rephosphorylated by viable myocytes during reperfusion to regenerate ATP stores. ^{14, 15} AMP and IMP nucleotides, however, are further degraded by 5'NT to adenosine and inosine nucleosides. ^{16, 17} These are freely diffusible, are washed out of the cytoplasm during reperfusion, and are consequently not available for resynthesis of ATP. With the use of pentoxifylline as an inhibitor of 5'NT in the adult heart in this study and in a previous study from this laboratory, ¹⁸ ATP was completely preserved. This suggests that nucleotide breakdown by 5'NT is an important mechanism of high-energy phosphate loss in the adult heart. ^{12, 19, 20}

5'NT activity has been reported to be 68% lower in neonatal hearts than in adult hearts, and this lower 5'NT activity has been correlated with superior postischemic recovery. ^{1, 9} If 5'NT is less important in causing high-energy phosphate breakdown in the neonatal heart, then than pharmacologic inhibition of 5'NT may provide little or no additional preservation of high-energy compounds and postischemic function. The finding in this study that 5'NT inhibition by pentoxifylline did not improve recovery of high-energy phosphates or function in neonates supports this hypothesis. This has implications for protection of both immature and mature heart muscle.

In neonatal hearts, Baker, Boerboom, and Olinger ⁴ showed that St. Thomas' Hospital cardioplegia at 14° C did not improve protection over hypothermia alone. In fact, cardioplegia was associated with a moderate loss in function compared with hypothermia without cardioplegia. Although hearts in the current study were kept at 34° C and are not strictly comparable, the findings that cardioplegia did not improve recovery of cardiac output, developed pressure, or high-energy compounds in the neonatal age group are in agreement with the concept that cardioplegia may be superfluous in the immature heart.

The general inefficacy of cardioplegia in neonates and the specific lack of protection of 5'NT inhibition suggests that the neonate degrades high-energy phosphate compounds during global ischemia via other pathways. One possible pathway is the conversion of ADP to deoxyadenosine diphosphate, which is used in the synthesis of DNA. ²¹ The extremely high rate of biosynthesis in the neonatal heart may therefore result in depletion of ATP precursors in a fashion that would be insensitive to 5'NT inhibition. Another possible mechanism of loss of high-energy phosphates in the neonatal heart is amino-acid synthesis. In lower organisms, histidine is synthesized through a series of reactions that begins with the condensation of ATP with 5-phosphoribosyl-1-pyrophosphate. This reaction is thought to be a molecular relic of an earlier evolutionary stage in which ATP was an essential substrate in both nucleic acid and amino-acid synthesis, as well as the major currency of energy exchange. ²² It is not known whether the immature heart retains the ability to support these reactions. In the context of the mechanisms of high-energy phosphate loss proposed herein, membrane depolarization with cardioplegia would have little effect on nucleic acid or amino-acid biosynthesis and would not be expected to contribute to myocardial protection.

The explanation for lower high-energy stores and poorer recovery of cardiac output and developed pressure in 1-month-old hearts with the addition of pentoxifylline to St. Thomas' Hospital cardioplegic solution remains speculative. It may be incorrect to explain this observation solely on the basis of 5'NT inhibition. Pentoxifylline has a variety of other effects, including antineutrophil activity, downregulation of cytokine production, increased deformability of red blood cells, and stimulation of prostaglandin synthesis. ²³ Pentoxifylline may act by a mechanism other than 5'NT inhibition in the 1-month-old heart, exerting an adverse effect. This may include the stimulation of ATP breakdown by alternative biosynthetic pathways.

One of the potential weaknesses of this study involves our use of crystalloid rather than blood in the perfusion circuit. Blood-perfused hearts have a very different hemodynamic profile from crystalloid-perfused hearts. As described by Walters and colleagues, ²⁴ functional and biochemical parameters at baseline and after ischemia are better with blood than crystalloid perfusate. We do not, however, believe that use of crystalloid to adjudicate the effect of pentoxifylline jeopardizes this study. In fact, it may strengthen the validity of our findings. Because pentoxifylline has a variety of hemorheologic and provocative effects on neutrophils and red cells, use of blood in the perfusion circuit might add confounding variables. For this reason we believed that it would not be possible to ascertain the effects of pentoxifylline on 5'NT activity in a blood-perfused model.

The activity of the 5'NT enzyme was not directly measured in this study. There is an assay for 5'NT that was recently used to show that there is lower 5'NT activity in the neonatal rabbit heart than in the adult heart. ¹ This assay is an enzyme kinetic method devised by Arkesteijn ²⁵ and based on the rate of decrease in absorbance observed with the oxidation of the reduced form of nicotinamide-adenine dinucleotide to nicotinamide-adenine dinucleotide, occurring as part of a series of linked reactions. For our high-energy phosphate determinations with high-performance liquid chromatography, tissue is lyophilized, and values are indexed by the dry weight of the sample. In contrast, tissue for the 5'NT activity assay cannot be desiccated, and values are expressed as nanomoles per minute per gram wet weight. Because of the small size of the neonatal hearts, there was not adequate tissue to divide the specimen and assign pieces to the different tissue preparations. In addition, when hearts in different age groups are compared, use of wet tissue weight to index enzyme activity is not ideal, inasmuch as the newborn heart has a greater water content than the adult heart. Furthermore, because hearts are edematous to a varying degree after ischemia and reperfusion, it may not be possible to compare activities of 5'NT with wet tissue weight used as the index. Direct measurement of 5'NT may be important, but the assay probably needs to be modified to make more precise comparisons.

The most important implications of this study are for the methods of myocardial protection used in adult hearts. These results indicate that 5'NT inhibition may permit the mature myocardium to behave like that of the neonate with respect to ATP metabolism. This in turn results in superior restoration of high-energy phosphate stores and enhanced cardiac function. Standard methods of myocardial protection during induced cardiac arrest may thus be augmented by pursuit of a neonatal strategy for avoidance of ischemic injury.

* Current address: Ara K. Pridjian, MD, The Ochsner Clinic, 1514 Jefferson Highway, New Orleans, LA 70121.

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Ara K. Pridjian Edward L. Bove Steven F. Bolling Flavian M. Lupinetti Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pridjian, A. K. Articles by Lupinetti, F. M. Search for Related Content PubMed PubMed Citation Articles by Pridjian, A. K. Articles by Lupinetti, F. M.