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J Thorac Cardiovasc Surg 1995;109:1155-1163
© 1995 Mosby, Inc.
CARDIOPULMONARY BYPASS, |
Winnipeg, Manitoba, Canada
Supported by the Medical Research Council of Canada (grant No. 91090P-21383-CEAA-15999).
Received for publication Jan. 28, 1994. Accepted for publication August 26, 1994. Address for reprints: Ganghong Tian, MD, PhD, Institute for Biodiagnostics, National Research Council Canada, 435 Ellice Ave., Winnipeg, Manitoba, Canada R3B 1Y6 (NRC 34755).
Abstract
This study was designed to assess the effects of intermittent warm blood cardioplegia on myocardial energy metabolites, intracellular pH, and contractile function. The isolated blood-perfused pig hearts were divided into three groups. After 30 minutes of control perfusion, the hearts in group 1 (n = 10) received 90 minutes of continuous warm (37° C) blood cardioplegia; the hearts in group 2 (n = 9) received six 5-minute periods of warm blood cardioplegia, interrupted by six 10-minute episodes of ischemia (37° C). The hearts were then reperfused for 30 minutes. The hearts in group 3 underwent 150 minutes of control perfusion without cardioplegia or ischemic episodes. Phosphorus 31-nuclear magnetic resonance spectra showed that a 10-minute interruption of warm blood cardioplegia decreased phosphocreatine levels and intracellular pH by approximately 47% (p < 0.01) and 0.12 unit (p < 0.05), respectively, and increased inorganic phosphate levels by approximately 87%, whereas resumption of cardioplegia for 5 minutes resulted in almost 100% recovery of phosphocreatine and inorganic phosphate levels and intracellular pH. More important, subsequent interruptions did not result in any cumulative changes in phosphocreatine level, inorganic phosphate level, or intracellular pH beyond those changes observed after the initial cardioplegic interruption. Moreover, during reperfusion there were no significant differences in adenosine triphosphate and phosphocreatine levels among the three groups of hearts. Furthermore, hearts from groups 1 and 2 showed comparable recovery of contractile function. These results indicate that six 10-minute interruptions and six 5-minute restorations of warm blood cardioplegia caused only mild and reversible changes in myocardial energy metabolites and intracellular pH and these changes were not cumulative. This study suggests that antegrade intermittent warm blood cardioplegia may provide as much myocardial protection as does antegrade continuous warm blood cardioplegia in the normal heart. (J THORACCARDIOVASCSURG1995;109:1155-63)
Continuous normothermic blood cardioplegia has recently emerged as an alternative method of myocardial protection. This method may, theoretically, be the optimal technique for heart protection because it aims to avoid myocardial ischemia and subsequent reperfusion injury. 
1 In practice, continuous infusion of warm blood cardioplegia may result in inadequate visualization of the operative field and make intracardiac manipulations difficult in some circumstances. 2 Therefore surgical precision may necessitate discontinuation of cardioplegia for short intervals. 2 The interruptions of warm blood cardioplegia will impose repetitive ischemic episodes on the "diseased" myocardium. Although a short period (<15 minutes) of ischemia is insufficient per se to cause severe myocardial injury, it is not clear whether recurrent ischemic episodes have a cumulative effect on myocardial metabolism and function, which may ultimately cause irreversible injury.
Experimental results in this context are conflicting Lange, Ware, and Kloner 3 found that short periods (5 and 15 minutes) of occlusion of the left anterior descending coronary artery resulted in significant depression of contractile function in the canine heart. These alterations, however, were not cumulative after three repetitive coronary artery occlusions. Reimer and associates 4 reported that one 10-minute period of ischemia caused a 61% loss of myocardial adenosine triphosphate (ATP) and a 41% loss of adenine nucleotides from the canine heart, whereas four 10-minute periods of ischemia did not cause further loss of ATP or adenine nucleotides. Other investigators have reported contradictory results. 5,6 It must be noted that the myocardial ischemia in the aforementioned experiments was regional and simply induced by occlusion of a coronary artery; it was not initiated by infusion of a cardioplegic solution. The changes in myocardial high energy metabolites and intracellular pH, as well as contractile function during reflow, may be expected to be significantly different in the heart in which myocardial ischemia is induced by intermittent warm blood cardioplegia.
The present study was done to determine the effects of intermittent warm blood cardioplegia on myocardial high energy phosphates, intracellular pH, and contractile function in the normal heart. The changes in myocardial energy metabolites (ATP, phosphocreatine, and inorganic phosphate) and intracellular pH were compared in pig hearts subjected either to 90 minutes of continuous warm blood cardioplegia or to six 5-minute periods of warm blood cardioplegia interrupted by six 10-minute periods of normothermic ischemia. Because myocardial adenine nucleotides and energy charge are restored within 3 minutes of reperfusion and recovery of phosphocreatine takes place rapidly on reperfusion, 5 minutes of reflow of cardioplegic solution was chosen. 7 A 10-minute interval of ischemia was used to mimic the clinical situation. Myocardial energy metabolites and intracellular pH were monitored with the use of phosphorus 31-nuclear magnetic resonance (31P-NMR) spectroscopy because this technique is noninvasive and nondestructive and a heart can serve as its own control. 8
MATERIAL AND METHODS
Isolated heart preparation
The pig heart was chosen as the animal model because it shares many similarities with the human heart. 9-11 All animals received humane care in compliance with the "Guide to the Care and Use of Experimental Animals" (first edition) formulated by the Canadian Council on Animal Care and the protocols were approved by the National Research Council Canada Animal Care Committee.
Domestic pigs that weighed 20 to 30 kg each were sedated with an intramuscular injection of diazepam (5 to 10 mg) and ketamine (25 mg). Anesthesia was maintained with 1% halothane in O2/N2 after tracheotomy. The respiratory rate and volume were adjusted to keep the arterial blood gas levels within the normal physiologic range. The brachiocephalic artery was cannulated at the level of the common carotid artery for arterial pressure monitoring, blood sampling, and infusion of cardioplegic solution.
A sternotomy was done. The brachiocephalic and subclavian arteries were dissected. The pericardium was opened longitudinally along the midline. The ascending aorta and the main pulmonary artery were isolated by threading umbilical tape around the origin of the descending aorta. Anticoagulation was provided by injection of heparin (3000 IU) into the superior vena cava. A cannula was inserted centrally in the brachiocephalic artery. The brachiocephalic artery, subclavian artery, and superior and inferior venae cavae were then clamped in succession. The descending aorta was clamped, and heparinized cold cardioplegic solution (10° C) was infused (10 ml/kg body weight) into the aortic root via the brachiocephalic arterial cannula. The right and left atria were cut to allow drainage of the cardioplegic solution and to prevent the warm blood in the lungs from returning to the heart. Cold Krebs-Henseleit (K-H) solution was used for topical hypothermia in the chest cavity because pig blood was collected for perfusing the heart.
The heart was then excised and immersed in cold cardioplegic solution. The brachiocephalic artery was joined to a cannula to be connected to a Langendorff perfusion apparatus. To measure left ventricular pressure a latex balloon was fixed in the left ventricle with a purse-string suture placed in the mitral valve and tied around the balloon mounting plug. Accumulation of blood in the left ventricle from thebesian flow was prevented by use of a small length of polyethylene.
A small glass ball filled with phenyl phosphonic acid was inserted into the right ventricle as a reference for the 31P-NMR signal intensities. Afterwards, hearts were placed in the magnet and perfused in the Langendorff apparatus (Fig. 1). The perfusion pressure was set at about 70 mm Hg at the beginning of the experiment and the corresponding flow rate was maintained throughout the protocol. It took approximately 20 minutes from cardiac in vivo arrest to reestablishment of coronary blood flow. Our previous studies showed that 20 minutes of ischemia at 12° C resulted in about a 20% decrease in phosphocreatine levels without notable alteration of ATP levels, which suggests that cardiac in vivo arrest during model preparation may not have a substantial impact on high energy phosphate stores. 12
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Perfusion solutions
During the control perfusion and reperfusion periods, the pig hearts were perfused with a mixture of autogenous blood and modified K-H solution (1:1, vol/vol) such that the hematocrit level was 12% to 15%. K-H solution had the following composition: NaCl 118 mmol/L, KCl 4.7 mmol/L, MgSO4 1.2 mmol/L, glucose 11 mmol/L, NaHCO3 25 mmol/L, CaCl2 1.75 mmol/L, ethylenediaminetetraacetic acid 0.5 mmol/L, KH2 PO4 1.2 mmol/L, dextran 2%, and bovine serum albumin 0.5%. The mixture was bubbled vigorously with a 95% O2 :5% CO2 gas mixture to a final pH of 7.5. Cardioplegic arrest was done with a 16 mmol/L K+ mixture of autogenous blood and K-H solution with a hematocrit level of 12% to 15%.
Assessment of contractile function
Heart rate, left ventricular developed pressure (left ventricular peak pressure minus end-diastolic pressure), and maximal rate of pressure increase (+dP/dt) and decrease (-dP/dt) were continuously recorded from a left ventricular balloon by means of a pressure transducer (model P23XL, Spectramed Inc., Oxnard, Calif.) during control perfusion and reperfusion with use of an electroencephalogram and polygraph data recording system (Grass model 79E, Grass Instrument Co., Quincy, Mass.). Contractile function was also assessed by calculating the product of developed pressure and heart rate to give the rate-pressure product.
Nuclear magnetic resonance spectroscopy
31P-NMR spectroscopy was done at 4.7 T on a Biospec spectrometer (Bruker, Karlsruhe, Germany) with a 40 cm horizontal bore magnet operating at a phosphorus frequency of 81.03 MHz. A home-built solenoid coil that surrounded the whole heart was used. Magnetic field homogeneity in the sample region was optimized by shimming on the sodium signal of the sample. Free induction decay (FID) signals were obtained by use of 4 K data points (10 K sweep width) and 45 degrees radio frequency single hard pulses. The pulse length and repetition time were 80 µsec and 1.5 seconds, respectively. Eighty FID signals were accumulated for each spectrum according to protocol. Thus each nuclear magnetic resonance spectrum was averaged over a 2-minute sample time. Accumulated FID signals were exponentially multiplied, resulting in 20 Hz line broadening, to improve the signal-to-noise ratio.
The observed phosphorus compounds included inorganic phosphate, phosphocreatine, and three peaks of ATP ( 
, ß , and 
peaks). The ß peak was used for quantifying ATP. Because absolute quantification of metabolites observed by nuclear magnetic resonance in a perfused organ is difficult, spectral data were expressed as the percentage of the initial ATP value that was obtained during control perfusion and set at 100%. Intracellular pH was calculated according to the chemical shift of the inorganic phosphate peak. 13
Protocol
Pig hearts were divided into three groups. After 30 minutes of control perfusion, hearts in group 1 (n = 10) were subjected to 90 minutes of continuous warm blood cardioplegia and hearts from group 2 (n = 9) underwent 90 minutes of intermittent warm blood cardioplegia, which consisted of six 10-minute episodes of ischemia (37° C) and six 5-minute periods of warm blood cardioplegia. After 90 minutes of "crossclamping," the hearts were then reperfused for 30 minutes. Hearts in group 3 (n = 4) were subjected to 150 minutes of blood perfusion as controls.
Statistical analyses
Statistical analyses were done with STATGRAPHIC version 5 software (STSC, Inc., Rockville, Md.). All results were expressed as the mean plus or minus standard error of the mean. A significant change in the level of phosphocreatine, inorganic phosphate, or intracellular pH during the period of intermittent cardioplegia was determined by use of one-way analysis of variance (ANOVA). In other words, ANOVA was used to determine whether or not intermittent warm blood cardioplegia would cause cumulative changes in phosphocreatine, inorganic phosphate, or intracellular pH. One-way ANOVA was also used to compare the values of ATP, phosphocreatine, and inorganic phosphate at the end of reperfusion among the three groups. Comparisons of contractile function among the three groups of hearts were made by one-way ANOVA with repeated measures. A significant difference was said to exist at a probability value of less than 0.05.
RESULTS
Effect of intermittent warm blood cardioplegia on myocardial energy phosphates
Representative spectra obtained from a heart subjected to intermittent warm blood cardioplegia are shown in Fig. 2. It is clear that interruption of cardioplegia resulted in a significant decrease in the intensity of the phosphocreatine peak with a corresponding increase in the inorganic phosphate peak. However, phosphocreatine and inorganic phosphate levels returned to normal during subsequent cardioplegic infusion. The changes in ATP, phosphocreatine, and inorganic phosphate levels obtained from the hearts subjected to intermittent warm blood cardioplegia are summarized in Fig. 3. ATP levels decreased slightly during 90 minutes of intermittent cardioplegia and recovered completely during reperfusion. Each interruption resulted in a large decrease in the phosphocreatine level and corresponding rise in inorganic phosphate level, whereas subsequent cardioplegic infusion resulted in complete recovery of the phosphocreatine and inorganic phosphate levels. The average values of phosphocreatine and inorganic phosphate obtained from nine hearts during control perfusion, reperfusion, and six interruptions are presented in Table I. Phosphocreatine level was 230% ± 10.3% and 265% ± 12.7% during control perfusion and reperfusion, respectively. Intermittent warm blood cardioplegia resulted in a significant drop in phosphocreatine level (p < 0.01). However, there were no significant differences (p > 0.05) between phosphocreatine values measured during the six interruptions. Moreover, although inorganic phosphate levels increased significantly (p < 0.01) during each interruption, the differences at the end of each of the six interruptions were not statistically significant (p > 0.05). The results suggested that under our experimental conditions intermittent warm blood cardioplegia did not result in a cumulative loss of phosphocreatine or gain in inorganic phosphate.
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Table I. Under our experimental conditions, intracellular pH during control perfusion and reperfusion was 7.22 ± 0.02 and 7.20 ± 0.03, respectively. The difference in pH between control perfusion and reperfusion was not significant (p > 0.05). A 10-minute interruption of warm blood cardioplegia resulted in an average decrease in intracellular pH by 0.12 unit. Subsequent interruptions did not result in further decreases in intracellular pH because there were no significant differences in intracellular pH at the end of each interruption (p > 0.05).
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This study showed that a 10-minute interruption of warm blood cardioplegia resulted in decreases in myocardial phosphocreatine levels and intracellular pH by 45% (p < 0.01) and 0.12 unit (p < 0.05), respectively, and an increase in inorganic phosphate levels by 88%, compared with their preischemic values. Five subsequent interruptions lasting 10 minutes each did not cause cumulative changes of these parameters. Furthermore, there were no significant differences in levels of myocardial energy metabolites (ATP, phosphocreatine, and inorganic phosphate), intracellular pH, or contractile function during reperfusion in the hearts that underwent either intermittent or continuous warm blood cardioplegia. The results suggest that intermittent warm blood cardioplegia with 10-minute ischemic intervals caused mild ischemic changes and these alterations were not cumulative, which indicates that warm blood cardioplegia may be safely interrupted in the normal heart for surgical precision without causing myocardial ischemic injury.
According to the levels of high energy phosphates, reversible myocardial ischemia has been arbitrarily divided into three phases: latency, survival time, and revival time. 14 During the first period of ischemia there are essentially no changes in the levels of ATP, phosphocreatine, or inorganic phosphate: oxidative phosphorylation is still the major energy source for maintenance of function and structure of the myocyte by using oxygen remaining in the myocardium in the form of oxymyoglobin, oxyhemoglobin, and physically dissolved oxygen. Myocardial oxygen consumption in the normal beating heart is about 10 ml/min per 100 gm tissue and the oxygen reserve in the myocardium at the onset of ischemia is about 1 to 2 ml/100 gm tissue. The latency period initiated by stopping coronary flow in the heart subjected to normothermic ischemia will last only 1 to 20 seconds. This period will be significantly prolonged in the cardioplegically arrested heart because oxygen consumption has been significantly reduced, from 10 to 1 ml/min per 100 gm. 14 As a result, the latency period in the heart subjected to intermittent warm blood cardioplegia is about 1 to 2 minutes. During this period, myocardial metabolism, structure, and function remain essentially unchanged.
During the second phase of ischemia (survival time), phosphocreatine is used to replenish ATP stores in the cytoplasm, which leads to a decrease in phosphocreatine level, accompanied by a rise in inorganic phosphate. This period ends when the phosphocreatine level decreases to 40% of its normal value and lasts about 1 to 3 minutes in the nonarrested heart. In contrast, Table I and Fig. 3 show that it took about 10 minutes for phosphocreatine levels to decrease to a similar level in the arrested hearts, which indicates that intermittent warm blood cardioplegia with 10-minute ischemic intervals resulted in only mild ischemic changes.
Additionally, one important effect of ischemia is the generation of protons derived from anaerobic glycolysis with breakdown of ATP and from other metabolic cycles that form protons, leading to a decrease in tissue pH. 15 The severity of ischemic injury has beenshown to be related to the extent of pH decrease. 16 Accumulation of protons can cause influx of Na+ and Ca++ via Na+/H+ and Na+/Ca++ exchange. More important, a fall in intracellular pH inhibits the activity of phosphofructokinase and consequently decreases energy production during ischemia. 15 It has been proposed that pH is an important factor in determining the difference between the effects of severe ischemia, which inhibits glycolysis, and mild ischemia, which accelerates glycolysis. 15 Conceivably, tissue pH is an important and reliable metabolic indicator of the magnitude of ischemic injury and of the adequacy of myocardial protection. The transition from mild acidosis to severe acidosis is rather arbitrary. It is generally accepted that intracellular pH below 6.2 represents severe acidosis. 13 In the present study, a 10-minute interruption of warm blood cardioplegia resulted in an intracellular pH decrease of only 0.12 unit (from a control value of 7.22 to 7.10) and subsequent interruptions did not cause further pH decreases (Table I). The intracellular pH at the end of each interruption remains within the normal physiologic range (Table I). Combined with the changes in the levels of myocardial phosphocreatine and inorganic phosphate, our results indicate that interruption of warm blood cardioplegia for 10 minutes repeated six times results in mild alterations in myocardial metabolic homeostasis, which soon recovers on resumption of coronary flow.
In addition, it is well known that reperfusion is not always fully beneficial although it is an absolute prerequisite for survival of the ischemic myocardium. 17-19 Under certain circumstances, reperfusion can result in additional deleterious effects in the ischemic myocardium. 17-19 A group of unwanted events consequent to reperfusion is defined as reperfusion injury, and the magnitude of reperfusion injury is closely related to the severity of ischemic injury. 17-19 As mentioned previously, the ischemic changes induced by six 10-minute interruptions of warm blood cardioplegia were mild and may not be sufficient to impose any significant damage on the myocardium. Under these conditions, recurrent restoration of cardioplegia may not cause any detrimental effect.
In addition, it has been documented that intracellular Ca++ overload is a critical mechanism responsible for reperfusion injury. 17-19 This Ca++ overload is, to a great extent, caused by a preceding ischemia-induced Na+ overload and by disruption of the sarcolemmal structure and dysfunction of the sarcoplasmic reticulum. Because the free energy of ATP hydrolysis ( 
GATP ) is normally about 15 to 20 kilojoules per mole greater than the energy required to drive the Na+/K+ pump and the decreases in ATP and intracellular pH during intermittent warm blood cardioplegia were small, the pump kinetics were not expected to be limited. 20 Thusintracellular Na+ would not increase significantly during intermittent warm blood cardioplegia although we did not measure intracellular Na+ during the course of our experiments. Therefore recurrent restoration of cardioplegia would not be expected to result in any increase in the level of intracellular Ca++. Furthermore, cardioplegia abolishes the action potential and inactivates myocardial Na+ and Ca++ channels. 14 This suggests that recurrent cardioplegia in our experimental pattern may not result in significant reperfusion injury. This was supported by our finding that hearts subjected to intermittent warm blood cardioplegia showed recovery of contractile function similar to that in those subjected to continuous cardioplegia.
Although ischemic metabolic alterations caused by intermittent warm blood cardioplegia were insubstantial and intermittent warm blood cardioplegia seemed to provide myocardial protection equal to that of continuous warm blood cardioplegia under our experimental conditions, we still do not know whether intermittent warm blood cardioplegia damages endothelial cells. In addition, 31P-NMR spectroscopy as used in this study provided only average values of energy metabolites and intracellular pH over the whole heart. It may be possible that some part of the myocardium was damaged (at a level below the detection sensitivity of our instrument) by interruption of warm blood cardioplegia because the myocardium in various parts of the heart may differ in susceptibility to ischemic insult. Additionally, the flow rate of intermittent cardioplegia is another important element in determining the efficacy or safety of intermittent warm blood cardioplegia because the distribution of cardioplegia solution between the subendocardium and the subepicardium can depend on the flow rate.
This study examined normal hearts of pigs that were perfused antegradely. Therefore the results obtained cannot be directly extrapolated to human hearts with significant coronary artery disease or hypertrophy. In addition, interruption of retrograde normothermic cardioplegia may have different consequences than interruption of antegrade normothermic cardioplegia. Furthermore, in situ hearts have noncoronary collateral flow that does not exist in the isolated heart preparation used in our studies. In conclusion, intermittent warm blood cardioplegia under our experimental conditions did not result in a cumulative severe ischemic injury and seemed to provide "equal" protection to that of continuous blood cardioplegia. However, its effect on the diseased and in situ heart requires further investigation.
Footnotes
From the Institute for Biodiagnostics,a National Research Council Canada, Winnipeg, Manitoba, Instituto di Clinica Cardiovasculare,b Cattedra di Cardiochirurgia, Ospedale S. Camillo De Chieti, Italy, and the Division of Cardiothoracic Surgery,c St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada. 
References
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