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Ann Thorac Surg 1996;62:1172-1179
© 1996 The Society of Thoracic Surgeons
Divisione di Cardiochirurgia, Spedali Civili; Salvatore Maugeri Foundation, IRCCS, Cardiovascular Pathophysiology Research Centre, Gussago; Cattedra di Cardiologia, Università degli Studi di Brescia; and Laboratorio Analisi, Opera Pia Paolo Richiedei, Gussago, Brescia, Italy
Accepted for publication May 30, 1996.
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Abstract |
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Methods. In the CWBC group, cardiac arrest was induced by continuous infusion of blood cardioplegia (10 mEq/L KCl) followed by 30 minutes of reperfusion with blood. In the IWBC group, after 5 minutes of perfusion with blood cardioplegia (10 mEq/L KCl), coronary flow was abolished for 10 minutes, followed by reperfusion with blood cardioplegia for 5 minutes. This sequence was repeated three times for a total period of 45 minutes. Finally the hearts were reperfused for 30 minutes with blood.
Results. Infusion of potassium induced a marked increase in coronary perfusion pressure (from 50 ± 3 to 98 ± 1 mm Hg; p < 0.01), which remained elevated throughout in the CWBC group, whereas in the IWBC group, it dropped to 0 during each no-flow period. In both groups, cardioplegia resulted in a significant reduction in oxygen consumption (from 5.5 ± 0.2 to 0.6 ± 0.03 mL O2•min-1•100 g-1 wet wt; p < 0.01). During CWBC, glucose extraction was significantly reduced (from 152 ± 10 to 64 ± 18 µg•min-1•g-1 wet wt; p < 0.01). Free fatty acid uptake and creatine kinase and lactate release were not affected. During IWBC, in contrast, a transient but significant release of creatine kinase (from 643 ± 254 to 2,234 ± 296 mU•min-1•g-1 wet wt; p < 0.01) and lactate (from 63 ± 22 to 374 ± 32 µg•min-1•g-1 wet wt; p < 0.01) occurred after each period of ischemia. Despite these metabolic differences, both cardioplegic procedures allowed a prompt and complete recovery of mechanical function and tissue content of high-energy phosphates.
Conclusions. Both CWBC and IWBC exert optimal protection in the isolated blood perfused rabbit heart. Thus, IWBC can be safely used to improve visualization of the surgical field.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentsAcknowledgmentsReferences |
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Hyperkalemic crystalloid hypothermic cardioplegia is the most commonly used technique to preserve the myocardium during cardiac surgery. Yet a number of authors have demonstrated both in human and animal studies the deleterious effects of hypothermia and ischemia, such as denaturation of proteins, swelling of cellular membrane, negative interference with energy production, and occurrence of oxidative stress [1–3]. All these alterations may lead to temporary impairment of mechanical performance (ie, stunning) in the postoperative period [3, 4].
Continuous warm blood cardioplegia (CWBC) has been recently proposed as an alternative cardioprotective procedure [5, 6]. Animal and human studies demonstrate that CWBC is superior to conventional cardioplegia, allowing greater postoperative metabolic and hemodynamic recovery [7, 8]. One limitation of CWBC is that during coronary operation, the continuous infusion of warm blood cardioplegia appears to disturb the operating field, making temporary interruption of the infusion necessary. This strategy, termed intermittent warm blood cardioplegia (IWBC), is gaining consent among cardiac surgeons, despite the absence of precise information on its metabolic and mechanical effects [9–11].
The aim of our study was to compare the protective effects of CWBC versus IWBC on energetic metabolism and mechanical performance of isolated and blood-perfused rabbit hearts.
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Material and Methods |
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Preparation of Blood Donor
Adult, male, white New Zealand rabbits (3.0 to 3.9 kg) maintained on a standard diet were used. They were anesthetized with sodium pentobarbital (30 mg/kg). Once tracheostomy was performed, the animals were mechanically ventilated on a positive pressure respirator (MD Industries, Mobile, AL). The respiratory rate was adjusted to keep the arterial oxygen tension, carbon dioxide tension, and pH at 80 to 90 mm Hg, 30 to 40 mm Hg, and 7.35 to 7.48, respectively, throughout the experiment. The right carotid artery was cannulated with a 13-gauge needle adapter to supply arterial blood to the isolated heart. A large-bore Tygon catheter was placed in the left jugular vein for delivery of venous blood from the isolated heart. Blood pressure was monitored via a catheter inserted into the left carotid artery and advanced into the thoracic aorta. To prevent coagulation of circulating blood, all animals were treated with 1,000 units/kg bolus of sodium heparin plus a 500 units/kg supplement every 60 minutes.
The perfusion circuit consisted of silicone tubing extending from the carotid artery of the support animal through a roller pump (model 1215; Harvard Apparatus, South Natick, MA) to a Teflon cannula encased in a water jacket heated to 37°C.
Preparation of Heart Donor and Perfusion of the Heart
Young, male, white New Zealand rabbits (0.8 to 1.0 kg) maintained on a standard diet were used. The rabbits received 500 units/kg of sodium heparin and were then stunned by a blow to the neck. The heart was rapidly removed and immersed in saline solution kept at room temperature and mounted on the perfusion apparatus within 1 minute. The isolated heart was placed in a glass water-jacketed chamber covered with plastic film. The chamber was siliconized with Sigmacote (Sigma Chemical Co., St. Louis, MO). The temperature of the chamber was kept constant at 37°C. The pulmonary artery was cut, allowing blood to drop by the force of gravity into the chamber from which the blood was delivered to the jugular vein of the support rabbit. Coronary perfusion pressure of the isolated heart was measured via a side arm of the perfusion cannula. The hearts were perfused with a perfusion pressure of 50 mm Hg. Pacing electrodes were placed in the right atrium, and the isolated heart was paced at 240 beats/min. Total coronary artery flow was measured by timed collection of blood from the chamber into a graduated cylinder.
The hearts were allowed to stabilize for at least 30 minutes before measurements were recorded.
Mechanical Performance of the Isolated Heart
To obtain an isovolumetrically beating preparation, a saline-filled latex balloon, connected via catheter to a Statham transducer (P 2306), was inserted into the left ventricle as previously described [12].
In separate experiments, measurement of left ventricular mechanical function was made before and after cardioplegia by generating function (Starling) and compliance curves. This was accomplished by measuring left ventricular developed and end-diastolic pressure on graded inflations of the intraventricular balloon.
Protocol
After stabilization, two groups were identified, each consisting of six separate experiments. In both groups cardioplegic arrest was induced by continuous administration of 25 mEq/L KCl with undiluted blood via a collateral arm of the perfusion cannula.
In the CWBC group, cardiac arrest was maintained for 45 minutes by continuous infusion of 10 mEq/L KCl with undiluted blood followed by 30 minutes of perfusion with blood without KCl. In the IWBC group, after 5 minutes of perfusion with blood cardioplegia, coronary flow was abolished for 10 minutes, followed by 5 minutes of reperfusion with blood plus cardioplegia (10 mEq/L KCl). This sequence was repeated three times for a total period of 45 minutes. Finally, the hearts were perfused for 30 minutes with blood without KCl.
At the end of the experiments, the hearts were frozen by clamping with aluminium tongs precooled in liquid nitrogen.
Assay of Cardiac Metabolism
Blood samples were taken simultaneously from the carotid artery of the blood donor rabbit and the pulmonary artery of the isolated heart for determination of oxygen, glucose, lactate, free fatty acids uptake, and creatine kinase (CK) release.
In the CWBC group, blood samples were collected 5 minutes before cardiac arrest; 15, 30, 45 minutes after cardioplegic infusion; and 30 minutes after the final KCl-free perfusion, before pressure-volume curve determination. In the IWBC group, blood samples were collected 5 minutes before cardiac arrest, 1 and 5 minutes after each "no-flow" phase, and 30 minutes after final KCl-free perfusion.
Glucose, lactate, FFA, and CK levels were measured using commercial kits from Boehringer according to the methods described by Deeg and associates [13], Wieland and Jagow-Westermann [14], Shimizu and colleagues [15], and Rosalki [16]. Myocardial oxygen consumption and high-energy phosphates were assessed as described elsewhere [17, 18].
Statistical Analysis
Data are expressed as the mean ± standard error. Statistical analyses between groups were performed by means of the unpaired t test. When several groups were compared, the data were first analyzed with a one-way analysis of variance followed by Tukey's test. A value of p less than 0.05 was regarded as significant.
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Results |
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Figure 1
shows typical mechanical traces from each group. The mean data of the six separate experiments are reported in Figure 2. Administration of a high dose of KCl (25 mEq/L) induced an immediate quiescence in both groups concomitant with an increase in coronary perfusion pressure (from 50 ± 0.5 to 95 ± 3 mm Hg; p < 0.01), which in the CWBC group remained elevated throughout KCl infusion. In the IWBC group, coronary perfusion pressure dropped to zero during each period of no flow, but increased again to the same levels as the CWBC group during each cardioplegic infusion (Fig 2B).
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). Figure 2 also shows coronary perfusion pressure returning to almost the baseline value after only 30 minutes of KCl-free perfusion in both groups.
The results of Starling curves are illustrated in Figure 3. Before cardioplegia, in the absence of diastolic stretching, optimal systolic pressure occurred at a volume of 0.3 mL. The same pattern was detected after cardioplegia, independently of the protocol employed.
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reports glucose and FFA uptake (Figs 4A, 4B), as well as lactate and CK release (Figs 4C, 4D). Before cardioplegic arrest, the isolated heart utilized glucose and FFA as preferential substrates; a small release of lactate and CK was also observed in this phase, despite normal oxygen utilization (5.3 ± 0.2 mL O2•min-1•100 g-1 wet wt) and pressure development (120 ± 3 mm Hg).
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). Free fatty acid uptake and lactate and CK release were unchanged. Subsequent perfusion with blood without KCl resulted in a prompt reestablishment of glucose utilization and no further changes in FFA uptake or in lactate or CK release. In the IWBC group, there was a significant increase in lactate and CK leakage 1 minute after each no-flow period (see Figs 4C, 4D). The release of these substances was transient, as 4 minutes after each reperfusion the values were not significantly different from those obtained before cardioplegia. During the final 30 minutes of KCl-free perfusion there was no further lactate and CK release. Glucose and FFA uptake remained constant throughout the experiment (see Figs 4A, 4B). During intermittent cardioplegia, glucose utilization was significantly higher with respect to that occurring in CWBC.
Table 1 shows tissue contents of high-energy phosphates as measured at the end of the experiments. Both techniques afforded optimal protection, as the values of each nucleotide were similar to those measured after 105 minutes of aerobic perfusion.
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Comments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentsAcknowledgmentsReferences |
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Experimental Model
We used an isolated and blood-perfused rabbit heart preparation, which is a relatively simple, reproducible, and inexpensive model closely mimicking the events occurring during cardiopulmonary bypass. Furthermore, this model has other advantages:
STABLE LOAD CONDITIONS.
Mechanical performance of the isolated heart is not affected by preload or afterload influence due to the peripheral changes that may occur during continuous infusion of blood cardioplegia in vivo [8].
OCCURRENCE OF ISCHEMIC DAMAGE.
The model is very sensitive to the deleterious effect of ischemia. Forty-five minutes of normothermic ischemia, without any form of protection, results only in 55% ± 5% recovery of mechanical function during reperfusion, with oxidative stress and metabolic damage [12], which makes this model ideal for testing the effects of cardioprotective agents.
UTILIZATION OF PHYSIOLOGIC SUBSTRATES.
The isolated heart is perfused with blood from a homologous donor, and can thus make use of all the physiologic substrate. In our experiments during the aerobic period the isolated heart utilized glucose and FFA as preferential substrates; oxygen consumption was similar to the value observed in an in vivo preparation under aerobic metabolism [17]. In our preparation, like in patients subjected to extracorporeal circulation, even under aerobic conditions the myocardial arterial-venous difference in lactate content may be negative. This could be explained by considering the ability of FFA at high concentration (1,234 ± 234 µmol/L) to block lactate utilization [19].
The most important limitation of the model is represented by the use of hearts with a normal coronary tree [20]. We cannot exclude that the presence of coronary stenoses may cause a maldistribution of the cardioplegic solution. Further, the absence of the pericardium rules out the potential role of collateral circulation during coronary flow arrest.
Experimental Protocol
We tried to mimic the procedure normally followed in humans. The amount of KCl used for the induction and maintenance of cardiac arrest is comparable with that reported in the literature [8]. The increase in kalemia, due to continuous infusion of cardioplegia, remained within the physiologic range. The duration and number of no-flow episodes is similar to that required to perform three or more distal anastomoses. Blood sampling was minimized to avoid a reduction in circulating volume.
Effects of Cardioplegia on Cardiac Mechanics
Data on CWBC are controversial. In animal models, CWBC has been shown either to provide the best preservation of left ventricular function [7], or to add no advantage, or even to cause a decrease in regional contractility of the myocardium distal from the coronary occlusion [21, 22]. The discrepancies are explained in terms of different via of cardioplegia infusion, coronary anatomy, and sensibility of the end points. Recent clinical studies confirm the superiority afforded by CWBC, but few studies have investigated the effects of flow interruptions [10, 11]. Our data show a complete recovery of systolic pressure after 3 minutes of KCl-free perfusion. This is also confirmed by the pressure-volume curves, which rule out the occurrence of stunning. Three cycles of flow interruption do not alter this pattern. These results have been recently borne out by Tian and associates [23] in a model of isolated pig heart perfused with a mixture of blood and Krebs-Henseleit solution. They found a complete recovery of positive rate of change of ventricular pressure during reperfusion after IWBC.
It is worth recalling that in our experimental model, KCl arrest plus 45 minutes of total hypothermic ischemia resulted in a transient left ventricular dysfunction on reperfusion (data not shown). In addition, infusion of KCl raises coronary perfusion pressure, which in a constant-flow perfusion model expresses an increase of coronary resistances. This is not surprising, as the depolarizing action of KCl on the voltage operator channels of the coronary artery smooth muscle cell, resulting in coronary constriction, is well known [24]. Our data also show that this phenomenon is reversible and unlikely to interfere with cardioplegia distribution in the coronary arteries, as shown by the optimal cardiac protection achieved.
Effects of Cardioplegia on Heart Metabolism
In the CWBC protocol, cardiac arrest subsequent to cardioplegia infusion resulted in reduced energy utilization by the heart, as implied by the reduced oxygen and glucose consumption. Our finding of unchanged myocardial content of high-energy phosphates hints that myocardial oxidative phosphorylation capacity was maintained throughout the experiment. Free fatty acids were the only substrate utilized by the heart during cardioplegia, which probably depends on the high circulating levels of FFA due to heparinization. Furthermore, glucose consumption increased immediately after the beginning of the KCl-free perfusion, revealing a perfect contraction-metabolism match.
Hearts subjected to IWBC behaved quite differently. Despite maintenance of quiescence, a reduction in contraction was not matched with a reduction in glucose uptake. For each reperfusion cycle, there was an abundant lactate release from the heart, likely supported by the maintained anaerobic glucose utilization. Tian and associates [23] have shown by using phosphorus-31 nuclear magnetic resonance that a similar period of interruption in warm blood cardioplegia results in a pH and CP decline, and in a concomitant inorganic phosphate increase. Such metabolic disarrangements are likely to activate anaerobiosis. Upon reperfusion we found a CK release, probably representing a wash-out phenomenon rather than real damage, as indicated by the prompt recovery of function by the heart. Despite this alteration in cardiac metabolism leading to a more pronounced anaerobic carbohydrate utilization in IWBC than in CWBC, the cardioprotection achieved was identical in the two groups. This is also confirmed by the complete preservation of energy charge measured at the end of the experiments.
It may well be that IWBC exerts cardioprotection by preconditioning the heart. It has been recently demonstrated that short periods of ischemia followed by reperfusion make the heart more resistant to subsequent ischemic insult, although the mechanisms of action of preconditioning remain unknown [25].
In summary, our data show that both CWBC and IWBC exert optimal protection, at least on the isolated, blood-perfused rabbit heart.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentsAcknowledgmentsReferences |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentsAcknowledgmentsReferences |
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentsAcknowledgmentsReferences |
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