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J Thorac Cardiovasc Surg 1995;110:103-110
© 1995 Mosby, Inc.

CARDIOPULMONARY BYPASS, MYOCARDIAL MANAGEMENT, AND SUPPORT TECHNIQUES

Role of sodium pump activity in warm induction of cardioplegia combined with reperfusion of oxygenated cardioplegic solution

Osaka, Japan

Received for publication May 26, 1994. Accepted for publication Nov. 15, 1994. Address for reprints: Tokumitsu Ko, MD, Department of Thoracic and Cardiovascular Surgery, Kansai Medical University, Fumizono-cho 1, Moriguchi City, Osaka 570, Japan.

Abstract

Na^+/K⁺adenosinetriphosphatase (sodium pump) may play a key role in the prevention of reperfusion injury caused by Ca²⁺overload. The present study was undertaken to investigate the role of sodium pump activity in warm induction of cardioplegia combined with reperfusion of oxygenated cardioplegic solution. Isolated and perfused rat hearts were subjected to 15 minutes of normothermic ischemia to produce a model of severely failing heart. The hearts then received myocardial preservation. Warm (37º C) or cold (4º C) oxygenated modified St. Thomas' Hospital solution was given for 5 minutes before and after 120 minutes of hypothermic cardioplegic arrest. Reduced myocardial pH during normothermic ischemia was adjusted toward the baseline level by administration of cold or warm oxygenated cardioplegic solution without a significant intergroup difference. Myocardial adenosine triphosphate levels decreased to less than 30% of the preischemic level during 15 minutes of normothermic ischemia, but were increased partly by induction of cold or warm oxygenated cardioplegia. Thus these metabolic indices failed to demonstrate the superiority of warm over cold oxygenated cardioplegia. Na^+/K⁺adenosinetriphosphatase activity in the membrane fraction was significantly stimulated by a cardioplegic dose of K⁺with maximum activity at 16 mEq/L. The enzyme activity of the heart measured after normothermic ischemia was reduced to less than 50% of that in the nonischemic heart. Although warm induction of cardioplegia and reperfusion of oxygenated cardioplegic solution maintained Na^+/K⁺adenosinetriphosphatase activity at the preischemic level, the enzyme activity was abolished at 4º C, which is the temperature used in cold cardioplegia. A subtoxic dose of ouabain (0.1 mmol/L) inhibited the enzyme activity of the heart undergoing this preservation regimen to approximately 50%. Warm induction and reperfusion of oxygenated cardioplegic solution showed significantly better recovery of isovolumic left ventricular function during reperfusion compared with that obtained with cold oxygenated cardioplegia. However, the beneficial effect of warm oxygenated cardioplegia on left ventricular function was compromised by inclusion of 0.1 mmol/L ouabain without a significant effect on myocardial metabolic parameters. These results suggest that stimulation of Na⁺pump activity may account for the beneficial effect of warm induction and reperfusion of oxygenated cardioplegic solution in the energy-depleted heart. (J THORACCARDIOVASCSURG1995;110:103-10)

Warm induction of cardioplegia combined with reperfusion of blood cardioplegic solution ("hot shot") has been accepted as an effective measure for myocardial preservation during heart operations, especially in patients with poor left ventricular function. ^1-3 The rationale of the hot shot is that increasing oxygen uptake by normothermia concomitant with maintaining cardiac arrest facilitates oxygen utilization toward cellular reparative processes rather than for unnecessary cardiac work. Although stimulation of cellular reparative processes appears to be an essential mechanism for enhanced myocardial preservation with warm blood cardioplegia, little information is available as to how warm blood cardioplegia operates on the damaged myocardial cells.

It appears that the enzyme systems that potentially participate in cellular recovery from ischemia and reperfusion injury are dependent on temperature and energy. In this regard, Na⁺/K⁺ adenosinetriphosphatase (ATPase) may represent a key enzyme in the prevention of myocardial reperfusion injury, because intracellular Na⁺ accumulation occurs as a result of Na⁺ pump inhibition concomitant with enhanced Na⁺ influx as a result of Na^+/H⁺ exchange during ischemia and reperfusion. ^4-8 Intracellular Na⁺ accumulation may then provoke massive influx of extracellular Ca²⁺ via Na⁺/Ca ²+ exchange, which gives rise to Ca²⁺ overload. ⁹ It is therefore anticipated that stimulation of this enzymeactivity by hot shot may reduce Ca⁺ induced reperfusion injury.

To test this hypothesis, we compared the effects of warm induction of cardioplegia combined with reperfusion of oxygenated cardioplegic solution on Na⁺/K⁺ ATPase activity and left ventricular function with those of cold induction and reperfusion of oxygenated cardioplegic solution in the failing heart model produced by severe but reversible ischemic damage before myocardial preservation. A subtoxic dose of ouabain, a modest inhibitor of Na⁺/K⁺ ATPase, was included in the warm oxygenated cardioplegic solution to substantiate an essential role of Na⁺/K⁺ ATPase activation in the efficacy of warm oxygenated cardioplegia. The results of this study suggest that stimulation of Na^+/K⁺ ATPase activity may represent a mechanism for enhanced myocardial preservation by warm cardioplegia.

METHODS

Perfusion techniques
Male Sprague-Dawley rats weighing between 250 and 350 gm were anesthetized by intraperitoneal injection with sodium pentobarbital (100 mg/kg). The chest was opened and sodium heparin (100 U/kg) was injected through the right atrial appendage. The heart was then removed quickly, placed in a nonrecirculating Langendorff perfusion circuit, and perfused with Krebs-Henseleit bicarbonate buffer solution equilibrated with 5% CO₂ and 95% O₂ with a pH of 7.4 at 37º C and at a constant perfusion pressure of 70 mm Hg. The Krebs-Henseleit bicarbonate buffer comprised (in millimoles per liter) NaCl 118, NaHCO₃ 25, KCl 4.6, KH₂ PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 2.5, and glucose 11. The isolated and perfused rat hearts were subjected to 15 minutes of normothermic ischemia to produce severe but reversible ischemic damage. The hearts then received cold or warm oxygenated cardioplegic solution with or without ouabain.

The first group of rat hearts received cold (4º C) oxygenated (5% CO₂ and 95% O₂ ) modified St Thomas' Hospital solution (cold oxygenated cardioplegia; COCP) for 5 minutes at a perfusion pressure of 70 mm Hg before (induction) and after (reperfusion) 120 minutes of hypothermic cardioplegic arrest. Hypothermic cardioplegic arrest was maintained by topical cooling and intermittent infusion of the same cardioplegic solution. Modified St. Thomas' Hospital solution comprised (in millimoles per liter) NaCl 95, NaHCO₃ 25, KCl 16, MgCl₂ 16, CaCl₂ 1.2, and glucose 11 at a pH of 7.4 at 37º C when equilibrated with a 5% CO₂ and 95% O₂ gas mixture.

The second group of rat hearts received warm (37º C) oxygenated modified St. Thomas' Hospital solution (warm oxygenated cardioplegia; WOCP) for 5 minutes at a perfusion pressure of 70 mm Hg before and after 120 minutes of hypothermic cardioplegic arrest, which was maintained as in the hearts with COCP.

The third group of rat hearts received warm oxygenated modified St. Thomas' Hospital solution containing a subtoxic dose of ouabain (0.1 mmol/L). Hypothermic cardioplegic arrest was introduced in the same manner as in the hearts with WOCP. These three groups of hearts underwent normothermic reperfusion with Krebs-Henseleit bicarbonate buffer solution for 25 minutes.

All 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 Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No 86-23, revised 1985).

Measurement of myocardial pH
Intramyocardial pH was measured with an ion-sensitive field-effective transistor pH sensor (model PH-2135, Kuraray Co., Ltd., Osaka, Japan) connected to a pH monitor (KR5000 pH/carbon dioxide tension monitor, Kuraray). Although the conventional technique of measurement of myocardial pH with a glass electrode requires calculation of the actual tissue pH by fitting the corresponding myocardial temperature and the calibration in vitro into the Nernst equation, ¹⁰ the ion-sensitive field-effective transistor pH sensor enabled on-line monitoring of temperature-corrected myocardial pH without a reference electrode.

Measurement of myocardial ATP
For analysis of myocardial ATP, the heart was freeze-clamped and lyophilized. Approximately 7 mg of the lyophilized sample was weighed and homogenized in 0.5N perchloric acid. The homogenate was centrifuged for 15 minutes at 1870 g and the supernatant was neutralized with 5N potassium hydroxide. The precipitate was removed again by centrifugation and the supernatant was neutralized with 5N potassium hydroxide. The resultant precipitate was removed by centrifugation and the supernatant was used for assay. Adenine nucleotide levels were measured by high-pressure liquid chromatography on a µ-Bondapak C-18 column (3.9 x 300 mm, Millipore Co., Bedford, Mass.) equilibrated with 0.1 mmol/L sodium phosphate buffer at pH 6.0 as described previously. ¹¹

Assay of Na⁺/K⁺ ATPase activity
The microsome fraction of the rat heart as the source of Na⁺/K⁺ ATPase was prepared as follows. Freeze-clamped myocardium (approximately 400 mg) was homogenized in ice-cold buffer solution containing 5 mmol/L imidazole-HCl (pH 7.0), 0.5 mmol/L ethylenediaminetetraacetic acid, 0.4 mmol/L phenylmethylsulfonyl fluoride, 4 µg/ml pepstatin, 4 µg/ml leupeptin, and 40 units/ml aprotinin (Trasylol) for three times 10 seconds with a Polytron homogenizer (Brinkman Instruments, Inc., Westbury, N.Y.). The homogenate was centrifuged at 10,000 g for 10 minutes, and the supernatant was spun at 20,000 g for 50 minutes in a Hitachi RP65 rotor (Hitachi Medical Corp., Tokyo, Japan). The pellets were suspended in 0.25 mol/L sucrose and stored at -80º C until use. Protein contents were determined by the method of Lowry and associates ¹² with bovine serum albumin used as a standard. About 15 µg of protein from the microsome fraction was preincubated in 0.1 ml of the standard medium containing 50 mmol/L imidazole-HCl (pH 7.0), 100 mmol/L NaCl, 16 mmol/L KCl, 4 mmol/L MgCl₂ , 1 mmol/L ethylenediaminetetraacetic acid, and 100 µg/ml saponin with or without 5 mmol/L ouabain at 37º C for 5 minutes. The reaction was initiated by addition of 2 mmol/L ATP. The incubation continued for 10 minutes and the reaction was terminated by cooling and addition of 0.05 ml cold 35% trichloroacetate. The inorganic phosphate released in the incubation medium was immediately measured according to a modification of the method of Parvin and Smith. ¹³ The Na⁺/K⁺ ATPase activity corresponds to the difference between the amounts of inorganic phosphate released in the absence and in the presence of ouabain into the incubation medium. The microsome fraction obtained from the hearts undergoing WOCP were also served for the study of the effect of 0.1 mmol/L ouabain on Na⁺/K⁺ ATPase activity, because ouabain bound with the enzyme during perfusion with WOCP containing ouabain may at least in part be lost during preparation of the membrane fraction so that the inhibitory effect of ouabain in vivo may not be determined accurately in vitro. Na⁺/K⁺ ATPase activity was expressed as micromoles per liter of inorganic phosphate per milligram of protein per hour.

Measurement of left ventricular function
So that isovolumic left ventricular pressure could be measured, a collapsed latex balloon, which was slightly larger than the rat's left ventricular chamber, was inserted into the left ventricle via a left atrial incision. The balloon was large enough that no pressure was generated by the balloon itself over the range of left ventricular volumes used in the experiments. The balloon was filled with bubble-free saline solution and attached to a pressure transducer connected to a multichannel recorder (Mingograf, Siemens-Elema, Division of Elema-Schönander, Inc., Solna, Sweden). The balloon volume was set to produce a left ventricular end-diastolic pressure (LVEDP) of approximately 10 mm Hg at the preischemic stage and this was kept constant throughout the experiment. A baseline measurement of left ventricular pressure was done after 15 minutes' maintenance of a hemodynamic steady state. At the end of the experiment a piece of the left ventricular muscle was weighed and heated (80º C) to dryness in an oven for 48 hours to determine the wet/dry weight ratio.

Statistical analysis
All data are presented as mean plus or minus the standard error of the mean. Statistical comparisons were done by an analysis of variance and Scheffe's multiple comparison test. p Values less than 0.05 were considered to be significant.

RESULTS

Effects of WOCP on myocardial pH
Myocardial pH decreased by 0.7 unit during 15 minutes of normothermic ischemia (Fig. 1). Induction of oxygenated cardioplegia for 5 minutes increased the myocardial pH level by about 0.45 unit, irrespective of whether the cardioplegic infusates were warm or cold and whether ouabain was included or not. Myocardial pH in these groups of hearts was almost unchanged during 120 minutes of hypothermic cardioplegic arrest, but gradually increased toward the baseline level during reperfusion.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Effects of warm induction combined with reperfusion of oxygenated cardioplegic solution on myocardial pH in energy-depleted rat heart. Squares, COCP (n = 8); circles, WOCP (n = 10); triangles, WOCP with 0.1 mmol/L ouabain in the solution (n = 8).

View larger version (27K): [in this window] [in a new window]   Fig. 2. Effects of warm induction combined with reperfusion of oxygenated cardioplegic solution on myocardial ATP in energy-depleted rat heart. Squares, COCP (n = 8); circles, WOCP (n = 8); triangles, WOCP with 0.1 mmol/L ouabain in the solution (n = 8).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effects of K+ concentrations on Na+/K+ ATPase activity in membrane fraction obtained from nonischemic rat heart. Each symbol represents mean plus or minus standard error of mean of at least eight experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Effects of warm induction combined with reperfusion of oxygenated cardioplegic solution on LVDP in energy-depleted rat heart. Squares, COCP (n = 8); circles, WOCP (n = 10); triangles, WOCP with 0.1 mmol/L ouabain in the solution (n = 8); **p < 0.01 versus COCP; #p < 0.05; ##p < 0.01 versus WOCP with ouabain in the solution.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Effects of warm induction combined with reperfusion of oxygenated cardioplegic solution on LVEDP in energy-depleted rat heart. Squares, COCP (n = 8); circles, WOCP (n = 10); triangles, WOCP with 0.1 mmol/L ouabain in the solution (n = 8); *p < 0.05; **p < 0.01 versus COCP; #p < 0.05; ##p < 0.01 versus WOCP with ouabain in the solution.

The present study demonstrated that warm induction of cardioplegia combined with reperfusion of oxygenated cardioplegic solution offered a significant benefit over hypothermic oxygenated cardioplegia for the recovery of left ventricular function during reperfusion in the energy-depleted rat heart. The beneficial effect of warm induction and reperfusion of blood cardioplegic solution has been discussed from the viewpoint of myocardial energy metabolism. ^{1, 14} The consistent finding of these studies is that myocardial aerobic metabolism is enhanced by warm blood cardioplegia. Similarly, our present study on myocardial energy metabolism showed rapid normalization of myocardial pH and a significant increase in ATP levels during warm induction of oxygenated cardioplegia. However, no recovery of myocardial ATP levels was noted during reperfusion with warm cardioplegic solution. Myocardial pH and ATP levels in the hearts undergoing COCP did not significantly differ from those in the hearts undergoing WOCP. This finding suggests that oxygenated cardioplegia even under hypothermic conditions is effective in maintaining aerobic energy metabolism. ^{15, 16} The recovery of myocardial pH and the level of myocardial ATP in this group of hearts during hypothermic cardioplegic arrest and reperfusion were comparable to those in the hearts with WOCP. Nevertheless, the hearts with COCP exhibited poorer recovery of left ventricular function after reperfusion. Such a discrepancy between myocardial energy metabolism and function has been explained by enhanced energy utilization probably for cellular reparative processes. ¹

The cellular event that requires energy in a temperature-dependent manner is the activation of various kinases and ATPases. We envisioned that Na⁺/K⁺ ATPase, among the enzymes involved in cellular homeostasis, plays a pivotal role in the prevention of reperfusion injury. Na⁺/K⁺ ATPase activity is an electrogenic process in which two Na⁺ extrude out of the cell while one K⁺ enters into the cell, thereby maintaining an appropriate transmembrane Na⁺ gradient. ¹⁷ Normally, Na⁺/K⁺ ATPase is a major energy-using process that accounts for as much as 40% of the basal metabolism of the body. ¹⁸ Further stimulation of theenzyme activity may be necessary when Na⁺ influx is increased under various pathologic conditions such as postischemic reperfusion. Myocardial reperfusion is known to promote massive Na⁺ influx by stimulating Na⁺/H⁺ exchange during the recovery from intracellular acidosis. ^{7, 8} Therefore loss of the enzyme activity under a hypothermic condition as demonstrated in vitro by our present study and those of others ^{19, 20} may allow Na⁺ accumulation and resultant Ca⁺ influx via Na⁺/Ca²⁺ exchange. ^{7-9, 21} Concomitant loss of pertinent regulation of intracellular Ca²⁺ levels during reperfusion leads to Ca²⁺ overload and subsequent cardiac dysfunction. ²²

The present study has indeed documented significant effects of K⁺, ischemic insult, and hypothermia on Na⁺/K⁺ ATPase activity. It has been shown that Na⁺/K⁺ ATPase activity is optimally stimulated at a K⁺ concentration of 20 mmol/L at physiologic pH and ATP levels. ²³ The measurement of intracellular Na⁺ activity in sheep heart Purkinje fibers has also revealed an inverse relation between external K⁺ and internal Na⁺/K⁺ ATPase concentrations ranging from 1 to 25 mmol/L. ²⁴ Our study confirms the previous work and suggests further that the enzyme activity may be suboptimum with perfusion with an oxygenated buffer solution containing normal K⁺, but that it can be stimulated by oxygenated cardioplegic solution containing a standard dosage of K⁺. The beneficial effect of potassium cardioplegic solution on intracellular Na⁺ activity has been demonstrated by Tani and Neely ²⁵ who showed that reperfusion with high K⁺ buffer reduced Na⁺ accumulation and C²⁺ uptake associated with better recovery of left ventricular function. On the other hand, the detrimental effect of hypoxia or ischemia on Na⁺/K⁺ ATPase activity has been documented by a number of investigators. ^26-28 Our measurements of Na⁺/K⁺ ATPase activity even in the optimized intracellular effectors, that is, neutral pH, no inorganic phosphate, and a normal cytosolic level of ATP, showed an approximately 50% reduction of the enzyme activity in the membrane fraction obtained from the hearts subjected to 15 minutes of normothermic ischemia. This extent of enzyme inhibition seems underestimated because myocardial ischemia produces acidic intracellular pH, accumulation of inorganic phosphate, and depletion of ATP, all of which are known to decrease Na⁺ pump activity. ⁵ Although the hearts that underwent WOCP and COCPmay have been provided optimum K⁺ and ATP levels to mimic those in the assay medium in vitro, virtually no Na⁺/K⁺ ATPase activity was noted under hypothermia. These results suggest that massive Na⁺ gain may occur during ischemia and hypothermic cardioplegic arrest.

The importance of the Na⁺ pump in preventing Ca⁺ induced reperfusion injury was substantiated by the use of ouabain. The concentration of ouabain (0.1 mmol/L) used in the present study was subtoxic and produced a positive inotropic effect without an increase in LVEDP in the nonischemic heart. An upward shift of LVEDP at constant left ventricular volume was considered an index of Ca²⁺ overload. ²⁹ Inclusion of 0.1 mmol/L ouabain in the solution for WOCP increased LVEDP during induction of WOCP, hypothermic cardioplegic arrest, and more prominently during reperfusion. The rise of LVEDP during reperfusion was associated with a significant decrease in LVDP compared with that in the hearts receiving WOCP in the absence of ouabain. These results can be interpreted as an inhibitory effect of ouabain against Na⁺/K⁺ ATPase activation exerted by WOCP. The approximately 50% reduction of Na⁺/K⁺ ATPase activity by 0.1 mmol/L ouabain in vitro supports this assumption.

The results of this study suggest that cold cardioplegia may predispose the heart to Ca²⁺-induced reperfusion injury by increasing Na⁺ gain as a result of Na⁺ pump inactivation. Our recent study that used the Ca²⁺ indicator fura 2 demonstrated that only a brief period of cold cardioplegia gave rise to a transient but significant increase in levels of intracellular Ca²⁺ at the moment of reperfusion. ³⁰ Such a harmful effect of hypothermia onintracellular Na⁺ and Ca²⁺ homeostasis may point to the appraisal of warm heart operation advocated by Lichtenstein and colleagues. ³¹ However, our results obtained from the crystalloid-perfused model may not simply be extrapolated to heart operations in which blood-perfused and reperfused systems are routinely used. The superiority of sanguineous perfusion over asanguineous perfusion has been demonstrated in isolated rat heart preparations. ³² Because blood is more potent in oxygen carriage and buffering capacity, differential responses to ischemia and reperfusion could be observed in animal and human hearts with blood cardioplegia. It is also possible that hormonal and humoral factors contained in the blood, such as thyroid and glucocorticoid hormones, modulate Na⁺/K⁺ ATPase activity. ³³ Nevertheless, it is reasonable to infer that high K⁺ concentrations, aerobic energy metabolism, and warm temperature are the common denominators for Na⁺/K⁺ ATPase activation and improved myocardial preservation in both warm oxygenated crystalloid and warm blood cardioplegia. Whether the efficacy of warm blood cardioplegia is related to the maintenance of intracellular Na⁺ and Ca²⁺ homeostasis remains to be investigated.

Footnotes

From the Departments of Thoracic and Cardiovascular Surgery^a and Pharmacology,^b Kansai Medical University, Osaka, Japan.

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