Developmental changes in reperfusion injury

J

THORAC CARDIOV\SC SCRG

1988;96:577-81

Developmental changes in reperfusion injury Comparison of intracellular ion accumulation in ischemic and cardioplegic arrest Developmental differences in ischemic and potassium cardioplegic arrest were evaluated in newborn (birth to 7 day old) and adult (6 to 12 month old) New Zealand white rabbit hearts isolated and perfused by Langendortrs method. An extracellular space washout technique was used to measure intracellular sodium and calcium in the two age groups after ischemia alone, after normothermic and hypothermic cardioplegia., and after cardioplegia with reperfusion. Although the intracellular ionic contents of nonreperfused adult hearts after 30 and 40 minutes of ischemia were identical, there was a twofold elevation in intracellular sodium level after 40 minutes of ischemia in the newborn hearts. Adult hearts arrested by normothermic potassium cardioplegia demonstrated no alteration in the intracellular ionic content. whereas in the newborn hearts, potassium cardioplegia produced excess intracellular calcium loading before reperfusion, which was greater than that occurring with ischemia alone. When hypothermia (12° C) was combined with cardioplegic arrest. a prereperfusion influx of sodium and calcium was not observed in the newborn hearts, and ionic reperfusion injury was blunted in both newborn and adult hearts. These studies demonstrate that the newborn heart is more susceptible than the adult to both ischemia and cardioplegia. This may be due to age-dependent differences in transmembrane passive diffusion, sodium/calcium exchange, or calcium slow channel properties and suggests alternative myocardial protective strategies for the newborn infant.

Ara K. Pridjian, MD, Sidney Levitsky, MD, Irvin Krukenkamp, MD, Norman A. Silverman. MD, and Harold Feinberg, PhD, Chicago. Ill.

h e immature heart thrives in the relatively hypoxic intrauterine milieu, supporting active myocellular growth and mitotic processes. In the early postnatal period. this capacity for hypoxic metabolism remains intact, but is lost with myocardial maturation. I The ability of the newborn heart to survive hypoxia is not understood, but may involve greater anaerobic glycolytic capacity or ability to perform de novo synthesis of high energy phosphate. ,·6 Hypoxia, ischemia, and cardioplegia represent different biochemical challenges to which newborn and adult hearts show different degrees of susceptibility. Whereas hypoxia involves continuous coronary perfusion with an From the Departments of Surgery and Pharmacology. University of Illinois College of Medicine at Chicago. Chicago. III. Received for publication Sept. 10.1987. Accepted for publication March 15. 1988. Address for reprints: Sidney Levitsky. \1D. Department of Surgery, Lniversitv of Illinois Medical Center. P.O. Box 6998, Chicago. IL 60680.

anaerobic solution, all flow is terminated in ischemia. With hypoxia, there is continuous supply of substrate and washout of acidotic metabolic byproducts. Conversely, with ischemia, the intravascular and interstitial spaces are stagnant pools, which are gradually depleted of substrate and buffering capacity. Cardioplegia preserves energy stores by producing rapid myocardial arrest, whereas energy is wasted by the paroxysmal. beating ischemic heart associated with noncardioplegic cross-clamping of the ascending aorta. Although the newborn heart is well-suited to both physiologic and experimental hypoxia, it tolerates ischemia and cardioplegia relatively poorly compared with the adult heart.':'? The potassium in cardioplegia provides myocardial arrest by depolarizing the transmembrane potential to -40 mY.II This change in potential has been demonstrated to affect the conductance properties of the voltage-dependent sodium and calcium channels, which allows intracellular flux of these ions down the electrochemical gradients. Although hypothermia and sponta577

The Journal of

578

Thoracic and Cardiovascular

Pridjian et al.

Surgery

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Fig. 1. Perfusion protocols for newborn (groups I. III. V. VII. IX. and XI) and adult (groups II. IV. VI. VIII. X. and XII) hearts.

neous ion-specific channel inactivation may retard these processes, even minimal transmembrane flux may result in significant pathologic intracellular ion accumulation during prolonged intraoperative arrest.'>" Intracellular accumulation of sodium and calcium are critical pathophysiologic features of the injured myocardium. Sodium exacerbates intracellular edema; calcium activates autolytic enzymes, depresses energy production, and produces myocardial contracture." In ischemic adult hearts, most of the accumulation occurs during reperfusion, when the energy-depleted acidotic postischemic cells are challenged with osmotic and oncotic pressure loads and the burden of resumption of contractile function." In a previous study, we'? focused exclusively on postischemic age-dependent differences in pathologic ion accumulation. In the current investigation, we determine the different effects of ischemia and of ischemia preceded by both normothermic and hypothermic potassium cardioplegia on intracellular sodium and calcium levels just before and after reperfusion to further elucidate developmental differences in the mechanisms of transmembrane ionic regulation.

Methods Hearts from newborn (birth to 7 day old) and adult (6 to 12 month old) New Zealand white rabbits were isolated and perfused by Langendorffs method. Details of this preparation have been reported previously." Briefly. after the induction of ether anesthesia, laparotomy was performed for heparin injection into the inferior vena cava. Hearts were excised and placed in a 4° C perfusate bath. and aortas were cannulated for coronary artery perfusion with vented polyethylene cannulas. Newborn and adult hearts were perfused at 37° C at 20 and 80 em H,O. respectively. A water-jacketed chamber sealed at both ends was used to maintain the hearts at 37° Cor \20 C. Each experimental group contained six hearts. After 30 minutes of equilibration. unprotected global ische-

mia was induced by terminating all perfusate flow for intervals of 30 minutes (Fig. I. groups I and II) or 40 minutes (groups III and IV). Cardioplegic arrest was accomplished with a 2-minute infusion of potassium cardioplegic solution at 37 or 12° C before terminating flow in newborn (groups V and IX) and adult (groups VI and X) hearts. Hearts were reperfused after 40 minutes of normothermic or hypothermic hyperkalemic arrest (groups VII. VIII. XI. and XII). The cellular cardioplegic solution previously described by our laboratory was made by the addition of 24 mEg potassium chloride and 7 gm glucose to I L of Ringer's solution." Trizma base (THAM) was added until the pH was 7.6. The solution was pumped through a 0.3 J.lm millipore filter before delivery. Sodium and calcium levels attributable to the intracellular space were measured with our modification" of the extracellular space washout technique of Alto and Dhalla." After variances were found to be homogeneous by Bartlett's test, one- and two-way analyses of variance were used to confirm the presence of differences between group means, The Tukey-Kramer test was then used to identify statistically different groups (p < 0.05).'0

Results After 30 minutes of normothermic ischemia, newborn and adult hearts had comparable intracellular sodium and calcium contents (Table I), which were not different from those of previously reported non ischemic hearts continuously perfused for 100 minutes.'? After 40 minutes of ischemia, sodium levels nearly doubled in newborn hearts, but remained within the normal range in adults (groups III and IV). Adult hearts exposed to 40 minutes of ischemia preceded by normothermic potassium cardioplegia showed no change in ionic content until reperfusion, whereupon calcium levels increased 208.8% and sodium levels increased 177.3% of nonreperfused values (groups VI and VIII). In the newborn hearts, the same conditions led to increases in both sodium and calcium (group

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Developmental changes in reperfusion injury

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579

Table I. The effects of 30 and 40 minutes of normothermic ischemia on intracellular sodium and calcium levels* in newborn and adult hearts Newborn

30 min ischemia, no reperfusion 40 min ischemia. no repcrfusion 40 min normothermic potassium cardioplegia. no repcrfusion 40 min normothermic potassium cardioplegia. 30 min repcrfusion 40 min hypothermic potassium cardioplegia. no repcrfusion 40 min hypothermic potassium cardioplegia. 30 min repcrfusion

Adult

Group

Calcium

Sodium

Group

Calcium

Sodium

I III V

5.16 ± 0.28 5.87 ± 0.46 8.77 ± 0.62t

64.78 ± 6.51 117.24 ± 10.30 121.16 ± 11.14

II IV VI

4.19 ± 0.27 4.07 ± 0.40 4.98 ± 0.29

62.80 ± 1.39 61.58 ± 6.24 53.51 ± 7.34

VII

10.40 ± 0.38

82.18 ± 6.12:1:

VIII

10.40 ± 1.12:1:

94.86 ± 8.85:1:

IX

5.05 ± 0.32

55.23 ± 6.63

X

5.45 ± 0.49

52.48 ± 5.48

XI

7.67 ± 0.51:1:

96.92 ± 8.50:1:

XII

8.26 ± 0.65:1:

56.98 ± 8.53

Each group contained 6 hearts. Normothermic ischemia was at 31' C: hypothermic ischemia at I ~. C. 'Expressed as micromoles per gram dry weight (mean ± standard error of the mean). tp

< 0.05 from 40-minute ischemic nonreperfused control values.

:j:p < 0.05 from nonreperfused cardioplcgic control values.

V), whereas reciprocal ionic changes were observed with reperfusion (group VII), with a net calcium influx and sodium efflux. Hypothermia at 12° C with potassium arrest prevented prereperfusion ion loading in newborn hearts (group IX). Newborn hearts accumulated both sodium and calcium if reperfused, whereas adults demonstrated an increase in calcium alone: Sodium and calcium levels were 175.5% and 151.9% of control values in newborns, respectively, whereas calcium levels rose to 151.6% of control values in adults (groups XI and XII). Discussion

The exact mechanisms of sodium and calcium accumulation in the damaged myocardial cell have not been defined, but probably involve enhanced activity of normal ion-specific membrane channels and passive diffusion through damaged sarcolemma." Early studies of ionic flux showed that the defects in ionic regulation are primarily due to enhanced influx and not to depressed efflux." Specifically, calcium may enter the damaged cell by sodium/calcium exchange, the calcium slow channel, and passive diffusion. Similarly, sodium influx may occur by sodium/calcium exchange, sodium fast channel flow, sodium flux through a damaged calcium channel, and passive diffusion. 22 The work of Shen and Jennings" showed that the injury that occurs during ischemia is not manifested until reperfusion. Using a method of flame photometry of regionally ischemic and reperfused canine myocardium, they noted that sodium and calcium accumulate

consequent to release of circumflex coronary artery occlusion." In addition, the studies of Nishioka, Nakanishi, and Jarmakani" of postischemic flux of radioactive ions into isolated rabbit ventricles perfused in a modified gamma counter also showed that most of the abnormal calcium influx occurs during the reperfusion interval. The results of our comparison of ischemia alone and ischemia with reperfusion using an extracellular space washout technique support these earlier studies (groups I, 11).10 To elucidate mechanisms of calcium influx occurring in the calcium paradox, Nayler and associates" used a perfusate formulation low in potassium to produce hearts with elevated intracellular sodium. These hypernatremic hearts, when satiated with calcium, demonstrated calcium influx in excess of that in hearts with normal sodium levels." Similarly, in our studies, the intracellular sodium level in the newborn heart is noted to be approximately twice normal after 40 minutes of ischemia (group III), and this rise in sodium level is followed by a massive calcium influx on reperfusion. 10 In this way, the "post-paradoxic" calcium level of Nayler and associates" and our postischemic calcium level in the newborn heart may both have sodium-dependent components that are due to sodium/calcium exchange. Reciprocal changes in sodium and calcium content noted with reperfusion after normothermic cardioplegia, though they did not attain statistical significance, may lend further support to a postinjury sodium/calcium exchange in the newborn heart (groups V and VII). Nayler and associates" also found a component of

The Journal of Thoracic and Cardiovascular Surgery

5 8 0 Pridjian et al.

post-paradoxic calcium influx that is sodium-independent. That our 30-minute ischemic/reperfused newborn hearts showed an increase in calcium levels while sodium levels remained normal is evidence that there is also a portion of postischemic calcium influx that is sodium-independent. As with the calcium paradox, the effects of sodiumloading in postischemic adult hearts has previously been examined. A similar profile of postischemic augmentation of calcium flux into sodium-laden hearts has been demonstrated and is probably also attributable to sodium/calcium exchange." However, we have no evidence that 40 minutes of ischemia actually produces sodium loading (group IX). For this reason, we question whether there is a large portion of pathologic calcium entry occurring by means of sodium/calcium exchange in the postischemic adult heart that is not sodium-loaded. If this pathway is not operative, then the majority of postischemic calcium influx must occur by the sodiumindependent passive or slow channel paths." Next, we used normothermic potassium cardioplegia to evaluate the effects of membrane depolarization on ionic fluxes. The membrane potential produced by the cardioplegic potassium concentration of 28 mmol/L is about -40 mV.!! At this potential, adult sodium fast channels are inactivated, sodium/calcium exchange has barely reached its reversal potential (favoring calcium influx over sodium efflux), and a trickle of calcium slow channel current is just detectable." 26. 27 It is not surprising, then, that there is no change in adult hearts after 40 minutes of potassium cardioplegia (group VI). Conversely, calcium elevation in the newborn heart in excess of that seen with ischemia alone was not expected (groups III versus V). These differences may signify a lower reversal potential for the sodium/calcium exchange protein or a lower activation threshold of the slow channel in the newborn heart. It is possible that these differences are due to immature forms of the channel or exchange proteins, which exist in greater quantity in newborn hearts. Our results corroborate age-dependent differences in slow channel and sodium/ calcium exchange processes noted by other investigators. 28-30 If the newborn heart does have slow channel and sodium/calcium activities at lower membrane potentials than the adult, it is likely that nondepolarizing agents such as magnesium would provide a safer myocardial arrest for the newborn infant than potassi-

urn." In the final series of experiments, we evaluated how the adjunct of hypothermia affects sodium and calcium fluxes in the potassium-arrested heart. Hypothermia provides a more rapid arrest than potassium alone and

lowers the myocardial metabolic rate to preserve high energy stores throughout the cardioplegic interval." Furthermore, hypothermia produces changes in membrane fluidity and ion-specific channel properties." 34 Thus the metabolic and membrane effects of hypothermia probably interact to maintain normal prereperfusion ionic contents in the newborn heart (group IX). Although calcium accumulation after reperfusion is not prevented, it is notably less than that with potassium alone (group VII versus XI). Previous studies have demonstrated the maintenance of normal functional and metabolic parameters in adult hearts after prolonged intervals of hypothermic cardioplegia and reperfusion." Yet, we found calcium accumulation in our preserved adult hearts (group XII), which suggests that calcium accumulation is probably not dependent on depressed energy stores. That this level of calcium influx is not seriously deleterious to myocardial function may be due to preservation of mitochondrial and sarcoplasmic reticular calcium sequestration. The metabolic inefficiency of the heart after cardioplegia, which we" have previously characterized, may be due to the energy required for these seqestration processes. We have demonstrated developmental differences in loss of ionic regulation after ischemia, cardioplegia, and hypothermic cardioplegia. These differences may represent distinct isogenic channel or exchange protein populations with different electrochemical properties in newborn and adult hearts. As controlled myocardial arrest in clinical cardiac operations involves manipulation of transmembrane ionic gradients mediated by these proteins, appreciation of the age-dependent differences is crucial to the design of more effective cardioplegic strategies for the newborn infant. REFERENCES I. Jarmakani JM, Nagatomo T, Nakazawa TA, Langer

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GA. Effect of hypoxia on myocardial high-energy phosphates in the neonatal mammalian heart. Am J Physiol 1978;235:H475-81. Dawes GS, Mott JC, Shelley HJ. The importance of cardiac glycogen for the maintenance of life in fetal lambs and new-born animals during anoxia. J Physiol 1958; 146:516-38. Mott .IC. The ability of young animals to withstand total oxygen lack. Br Med Bull 1952:17:144-8. Zimmer HG. Trendelenburg C, Kammermeier H, Gerlach E. Acceleration of adenine nucleotide synthesis de novo during development of cardiac hypertrophy. J Mol Cell Cardiol 1972;4:279-82. Zimmer HG, Trendelenburg C, Kammermeier H, Gerlach E. De novo synthesis of adeninenucleotides in the rat: acceleration during recovery from oxygen deficiency. Circ Res 1973:32:635-42.

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6. Jarmakani JM, Nakazawa TA, Nagatomo T, Langer GA. Effect of hypoxia on mechanical function in the neonatal mammalian heart. Am J Physiol 1978; 235:H475-81. 7. Opie JC, Taylor G, Ashmore PG, Kalousek D. "Stone heart" in a neonate. J THORAC CARDIOVASC SURG 1981; 81:459-63. 8. Bull C, Stark J. Cardioplegia in pediatric cardiac surgery: repair in the first year of life. In: Engelman RM, Levitsky S, eds. A textbook of clinical cardioplegia. Mount Kisco, New York: Futura, 1982:349-63. 9. Bull C, Cooper J, Stark J. Cardioplegic protection of the child's heart. J THORAC CARDIOVASC SURG 1984;88:28793. 10. Pridjian A, Levitsky S, Krukenkamp I, Silverman N, Feinberg H. Developmental changes in reperfusion injury: a comparison of intracellular cation accumulation in the newborn, neonatal, and adult heart. J THORAC CARDl0VASC SURG 1987;93:428-33. II. Raffa J, Mavroudis C, Trunkey DD, Ebert PA. Recovery of cardiac intracellular membrane potentials after potassium cardioplegia and hypothermia. Surg Forum 1977; 28:224-6. 12. Brown AM, Akaike N, Tsuda Y, Morimoto K. Ion migration and inactivation in the calcium channel. J Physiol (Paris) 1980;76:395-402. 13. Lee KS, Marban E, Tsien RW. Inactivation of calcium channels in mammalian heart cells: joint dependence of membrane potential and intracellular calcium. J Physiol (Land) 1985;364:395-411. 14. Lux HD, Brown AM. Single channel studies on inactivation of calcium currents. Science 1984;225:432-4. 15. Josephson IR, Sanchez-Chapula J, Brown AM. A comparison of calcium currents in rat and guinea pig ventricular cells. Circ Res 1984;54: 144-56. 16. Nayler WG. Calcium and cell death. Eur Heart J 1983; 4(suppl):33-41. 17. Shen AC, Jennings RB. Myocardial calcium and magnesium in acute ischemic injury. Am J Pathol 1972;67:41740. 18. Rao KS, Levitsky S, Holland C, Feinberg H, Fialkowski W, Merchant F. Does potassium-chloride-induced cardiac arrest protect the myocardium during aortic cross-clamping? Surg Forum 1976;27:262-4. 19. Alto LE, Dhalla NS. Myocardial cation contents during induction of calcium paradox. Am J Physiol 1979; 237:H713-9. 20. Sokal RR, Rohlf JF. Biometry. 2nd ed. New York: WH Freeman, 1981. 21. Nishioka K, Nakanishi T, Jarmakani JM. Effect of ischemia on calcium exchange in the rabbit myocardium. Am J Physiol 1984;247:HI77-84. 22. Chapman RA, Rodrigo GC, Tunstall J, Yates RJ, Busslein P. Calcium paradox of the heart: a role for

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