Ann Thorac Surg 2001;72:1964-1969
© 2001 The Society of Thoracic Surgeons
Original article: cardiovascular
Esmolol and cardiopulmonary bypass during reperfusion reduce myocardial infarct size in dogs
Hans J. Geissler, MD*a,
Karen L. Davis, PhDa,
L. Maximilian Buja, MDb,
Glen A. Laine, PhDc,
Michael L. Brennan, BSa,
Uwe Mehlhorn, MDa,
Steven J. Allen, MDa
a Department of Anesthesiology, University of Texas-Houston Medical School, Houston, Texas, USA
b Department of Pathology, University of Texas-Houston Medical School, Houston, Texas, USA
c Michael E. DeBakey Institute, Texas A&M University, College Station, Texas, USA
Accepted for publication July 20, 2001.
* Address reprint requests to Dr Geissler, Department of Cardiothoracic Surgery, University of Cologne, Joseph-Stelzmann-Str 9, 50924 Cologne, Germany
e-mail: hans.geissler{at}medizin.uni-koeln.de
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Abstract
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Background. Infarct size can be reduced by ß-blockade in acute myocardial ischemia. However it is unknown whether myocardial salvage is still effective when ß-blockade is limited to reperfusion.
Methods. After initiation of cardiopulmonary bypass, 20 dogs were submitted to 2 hours of regional left ventricular ischemia, followed by 2 hours of reperfusion. In 11 dogs ß-blockade was started with the onset of reperfusion (esmolol group). The remaining dogs received no treatment (control, n = 9). Infarct size was determined by tetrazolium chloride staining. Myocardial water content (MWC) and ultrastructural damage (electronmicroscopy) were determined from transmural biopsies.
Results. Infarct size was significantly smaller in the esmolol group compared with control (49% versus 68%, p < 0.05). After 2 hours ischemia there was no difference in MWC between groups, whereas after 2 hours reperfusion MWC of ischemic myocardium was significantly lower in the esmolol group than in the control (p < 0.05). Ultrastructural changes were typical for ischemia-reperfusion injury in both groups.
Conclusions. ß-Blockade may be cardioprotective during reperfusion through various mechanisms and may enhance myocardial salvage, even when treatment is initiated as late as with the onset of reperfusion.
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Introduction
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Therapeutic strategies in acute myocardial infarction are primarily aimed at reduction of myocardial oxygen consumption, prevention of ventricular arrhythmias, and immediate revascularization by thrombolysis, percutaneous transluminal coronary angioplasty or coronary artery bypass grafting (CABG). If revascularization is successful, alleviation of reperfusion injury appears to be essential for myocardial salvage. The ß-blocker esmolol has been shown to enhance myocardial salvage in a canine model of ischemia and reperfusion [1]. Myocardial infarct size was also found to be reduced in dogs when intravenous esmolol and percutaneous cardiopulmonary bypass (CPB) were combined [2]. However, clinical applicability of these models appears to be limited, as esmolol was administered continuously from a time before the onset of ischemia throughout the complete ischemic and reperfusion period, a protocol that does not resemble the clinical situation encountered in most patients. Frequently there is a considerable delay between the onset of ischemia and the initiation of treatment, because of various factors such as distance from treatment centers, verification of diagnosis, and others. It is unclear whether the beneficial effects of esmolol with regard to myocardial salvage can still be achieved when treatment is begun at a later time. However, evidence suggests that ß-blockade may be cardioprotective during reperfusion through various mechanisms [3, 4]. The purpose of this study was to determine if myocardial salvage can be enhanced if the esmolol administration is limited to the reperfusion period. In an effort to maximize the effect of ß-blockade we further chose a model of intracoronary administration of high-dose esmolol combined with CPB.
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Material and methods
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Animal preparation
All procedures were approved by The University of Texas Animal Welfare Committee and were consistent with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (7th ed., Washington DC: National Academy Press, 1996). Twenty mongrel dogs (32.2 ± 1.1 kg) of either sex were anesthetized by intravenous administration of thiopental sodium (25 mg/kg body weight [BW]), intubated endotracheally, and ventilated mechanically with 100% oxygen using a volume-cycled respirator (Siemens-Elema AB, Sundbyberg, Sweden). Anesthesia was maintained with intravenous infusion of 1% thiopental sodium in Ringer’s solution. Fluid-filled catheters were placed into the left femoral artery and vein for arterial pressure monitoring, arterial blood sampling, and fluid administration, respectively. We placed a 7F thermodilution catheter through the left jugular vein into the pulmonary artery for pressure and cardiac output measurements. The pressure monitoring catheters were connected to calibrated pressure transducers (Isotec, Healthdyne Cardiovascular, Irvine, CA), and data were recorded on a computer (MacLab, WorldPrecision Instruments, Sarasota, FL). A 7F catheter was introduced through the right jugular vein into the coronary sinus for coronary sinus blood sampling. The right femoral artery was exposed for subsequent CPB cannulation. A median sternotomy and pericardiotomy were performed.
Cardiopulmonary bypass
For systemic anticoagulation heparin was administered intravenously (250 IU/kg BW) followed by additional doses of 100 IU/kg BW given every 60 minutes throughout the experiment. Cardiopulmonary bypass was performed using a 14F arterial perfusion cannula placed into the prepared right femoral artery and a two-stage venous cannula (36F x 46F) placed into the right atrium and inferior vena cava. The left ventricle (LV) was vented with a 12F catheter inserted via the left atrium. We placed a regular cardioplegia cannula (Cobe Cardiovascular Inc, Arvada, CO) for measurement of aortic root pressure and aortic root perfusion, respectively, in the ascending aorta. The extracorporeal circuit and the hollow fiber oxygenator (Plexus, Sorin Biomedical, Irvine, CA) were primed with 1,200 mL of Ringer’s solution with 2% hetastarch (McGaw Inc, Irvine, CA) and 1,000 IU heparin. We used four roller pumps (Mod. 7000, Sarns Inc, Ann Arbor, MI) for extracorporeal circulation, cardiotomy suction, LV drainage, and aortic root perfusion, respectively.
Experimental protocol
After animal preparation and instrumentation, baseline measurements of all parameters were taken. Thereafter, CPB was initiated. Systemic arterial pressure was kept at 70 mm Hg for the duration of CPB. Regional LV ischemia was induced by tightening a vascular loop placed around the proximal third of the left anterior descending branch (LAD) of the left coronary artery. To avoid ventricular fibrillation we administered lidocaine intravenously at a dose of 1 mg/kg. After 2 hours of regional LV ischemia, the vascular loop snare was released. This time was marked as the onset of reperfusion. With the beginning of reperfusion we cross-clamped the aorta and started aortic root perfusion with oxygenated normothermic CPB blood and esmolol at a dose of 2 mg/min for 2 hours (esmolol group, n = 11). In the remaining dogs (control group, n = 9) the aortic root was perfused with oxygenated normothermic CPB blood without esmolol for 2 hours. No cross-clamp was performed in the control group. Aortic root pressure was measured at the aortic root cannula and was kept at 70 mm Hg in both groups. Perfusate temperature was monitored continuously by a temperature probe in the arterial line and kept at 37°C. Arterial and coronary sinus blood samples and transmural myocardial biopsy samples were taken at 120 minutes of ischemia and 120 minutes of reperfusion (Table 1).
Myocardial edema
Myocardial water content (MWC) was measured from transmural myocardial biopsy samples using a microgravimetric technique as described previously [5]. Briefly, a linear density gradient was prepared in a gradient former from two different mixtures of kerosene and bromobenzene, which were adjusted to a specific gravity of 0.990 and 1.080, respectively. Calibration of the gradient was performed using various K2SO4 solutions with known specific gravities and by recording the equilibration depth of 10-µL drops of the various solutions. Linearity of the gradient was confirmed by linear least-square regression analysis after plotting the equilibration depths versus specific gravity. The mean correlation coefficient was 0.995 ± 0.00036 (n = 20). To determine the specific gravity of myocardium we obtained full-thickness transmural biopsy samples using a commercially available biopsy needle (14G, Gallini Medical Products, 41037 Mirandola, Italy). These samples were placed into the density column and the equilibration depth was recorded after 1 min. Myocardial water content can be calculated using the equation:
 | ((1)) |
where SGmyo and SGdry are the specific gravities of the myocardial tissue and of dry myocardium, respectively. On conclusion of the experiment, the dog was euthanized with an intravenous overdose of pentothal and saturated potassium chloride. After a last myocardial tissue density measurement, the heart was rapidly excised. Approximately 2 cm2 of LV posterior wall was removed and dried at 60°C in an oven to a constant weight. The remainder of the heart was used for infarct size analysis (see below). SGdry was calculated using the following equation:
 | ((2)) |
where W and D are the wet and dry weights of myocardial tissue. We assumed that SGdry did not change over the experimental period. Measurements were performed in duplicate and taken at baseline, 120 minutes of ischemia, and 120 minutes of reperfusion. At each time biopsy samples were taken from ischemic (anterior LV wall) and nonischemic myocardium (posterior LV wall). As CPB and hemodilution are known to result in a mild increase of MWC, we calculated myocardial water gain in the ischemic area as the difference between MWC in ischemic and nonischemic myocardium, thereby correcting for changes induced by the experimental set-up and not by ischemia-reperfusion.
In 7 dogs (3 esmolol and 4 control animals), thin sections for electron microscopy were cut from myocardial biopsy samples. For this purpose, transmural biopsy samples were divided into subendocardial and subepicardial sections before fixation. Thin sections were analyzed in a blinded fashion for myocyte injury and intracellular edema using a semiquantitative score (Table 2).
Infarct size analysis
After 2 hours of reperfusion, the LAD was resnared and 1% gentian violet at a dose of 5 mL/kg was injected into the CPB reservoir. Gentian violet stained all tissue with the exception of myocardial tissue supplied exclusively by the LAD, thereby delineating the myocardial area at risk for infarction and accounting for variations in coronary anatomy. After euthanization of the dog, rapid heart excision and harvest of approximately 2 cm2 from the LV posterior wall for wet/dry ratio determination, the LV was sliced in 5-mm sections along the longitudinal axis, starting from the apex. The myocardial slices were incubated in 2% 2,3,5-triphenyl-2H-tetrazolium chloride (TTZ) for 15 min at 37°C. TTZ reacts with dehydrogenase enzymes in vital cells resulting in a brick red stain. Nonvital cells remain pale. Thereafter, slices were reincubated and fixed in 10% formalin. The outlines of unaffected myocardium (stained violet), ischemic myocardium without infarction (no violet stain, red TTZ stain), and infarcted myocardium (no stain) were traced manually onto acetate. Morphometric determination of unaffected, ischemic, and infarcted myocardium was performed using a digital planimetry software (Brain 1.4 for Macintosh). Infarct size was determined as the ratio of area at risk to infarcted area to correct for differences in coronary anatomy.
Statistical analysis
Data are presented as mean ± standard error or percentages. Myocardial water content data from the esmolol and control groups were compared using two-way analysis of variance for repeated measures. Post hoc comparisons were performed using paired Student’s t test with Bonferroni correction for two comparisons. An unpaired two-tailed Student’s t test was applied for comparison of infarct size and a Mann-Whitney U test for data of ultrastructural analysis. A p value of less than 0.05 was considered significant. Statistical analysis was performed using the Statistica 4.0 software package (StatSoft, Tulsa, OK).
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Results
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Hemodynamic and CPB parameters
Baseline measurements of cardiac index, mean arterial pressure, central venous pressure, and mean pulmonary artery pressure showed no difference between groups. After baseline measurements the animals were placed on CPB and mean arterial pressure was kept at 70 mm Hg for the remaining experiment. Table 3 shows hemodynamic and CPB relevant data.
Infarct size analysis
There was no difference between groups for the extent of area at risk relative to LV size (area at risk esmolol 35% ± 3.8% versus control 33.2% ± 5.6%). However, infarct size, determined as the ratio of infarcted myocardium to myocardium at risk, was significantly smaller in the esmolol group than in the control group (48.7% ± 4.6% versus 68.3% ± 4.7%; Fig 1).
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Fig 1. Infarct size (infarcted area/area at risk) was significantly smaller in the esmolol group compared with controls. *p < 0.05.
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Myocardial water gain of ischemic myocardium
No significant difference was noted between groups at base line and at 120 minutes of ischemia. At 120 minutes of reperfusion myocardial water gain of ischemic myocardium was significantly higher in the control group compared with the esmolol group (1.6% ± 0.2% versus 0.5% ± 0.3%; Fig 2).
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Fig 2. Myocardial water gain in ischemic myocardium. At baseline and 2 hours of ischemia no significant difference between groups can be seen. At 2 hours of reperfusion animals in the control group gained significantly more water in ischemic myocardium than animals in the esmolol group. *p < 0.05.
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Ultrastructure
Biopsy samples from both groups showed ultrastructural changes typical for ischemia and reperfusion injury. In both groups endomyocardial sections showed significantly greater cell damage in comparison with epimyocardial sections and the degree of cell damage was greater at 2 hour of reperfusion than at 2 hour of ischemia (Table 4). However, the differences between the groups were not significant.
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Comment
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Our data show significant reduction of infarct size with intracoronary administration of esmolol during reperfusion in unloaded dog hearts. Reduction of infarct size was associated with significantly less edema accumulation in ischemic myocardium during reperfusion.
Reduction of myocardial workload or unloading of the heart by circulatory support has been identified as an effective mechanism for alleviation of ischemia reperfusion injury [6]. The primary mechanism for the beneficial effect of circulatory support on myocardial salvage is reduction of myocardial oxygen consumption. Another mechanism to lower myocardial oxygen consumption is the reduction of heart rate and myocardial inotropy by ß-blockade. Because of its short half-life of approximately 9 minutes, the ß-blocker esmolol has been favored for the treatment of acute myocardial ischemia and unstable angina [7]. Experimentally, myocardial infarct size was reduced by esmolol, either when applied in combination with circulatory support or by ß-blockade alone [1, 2]. However, in distinction to these prior investigations in which esmolol was administered continuously from before the onset of ischemia, esmolol administration was limited to reperfusion in the present study.
Various mechanisms, other than reduction of myocardial oxygen consumption, have been suggested for the cardioprotective properties of ß-blockade. Measures aimed at augmentation of coronary blood flow during reperfusion, such as coronary retroperfusion, have been shown to limit myocardial necrosis in acute ischemia [8]. Coronary blood flow has also been found to be increased by ß-blockade, thereby improving myocardial oxygen supply and alleviating the severity of coronary stenosis [9]. Intracoronary administration of high-dose esmolol resulted in a significant decrease of coronary vascular resistance and increase of coronary blood flow [10]. In this study we found during reperfusion a significantly higher coronary sinus oxygen saturation in the esmolol group in comparison with controls, indicating a higher ratio of oxygen supply over oxygen demand in the esmolol group.
Metabolic changes induced by ß-blockade may also contribute to the cardioprotective effects. Myocardial lactate extraction and consumption during ischemia were significantly reduced by intracoronary infusion of esmolol in comparison with untreated animals [11]. Free radical–mediated injury induced by ischemia was also shown to be alleviated by esmolol [4]. Whereas the levels of oxygen radical scavengers, such as glutathione and superoxide dismutase, were significantly elevated in esmolol-treated animals, the concentration of malondialdehyde, a marker of lipid peroxidation, was markedly decreased. ß-Blockade may also exert cardioprotective effects by limiting the release of free fatty acids during myocardial ischemia. Increased plasma levels of free fatty acids have been shown to trigger ventricular arrhythmias and to impair activity of Ca2+ pumps in sarcoplasmic reticulum [12, 13]. As the increased release of free fatty acids during myocardial infarction is a predominantly norepinephrine-mediated response, this reaction may be attenuated by ß-blockade [14].
Myocardial reperfusion injury is associated with substantial intracellular and interstitial edema formation. Whereas only a mild swelling of cardiac muscle cells and no interstitial fluid accumulation is noted during myocardial ischemia, marked intracellular and interstitial edema develops rapidly during reperfusion [15]. In the present study, dogs treated with esmolol during reperfusion had significantly less edema in ischemic myocardium than controls. However, ultrastructural analysis of cardiac myocyte injury and intracellular edema showed no difference between groups. Therefore, we conclude that the difference in water content of ischemic myocardium was most likely a result of greater interstitial fluid accumulation in the control group. Interstitial edema may aggravate reperfusion injury by increasing oxygen diffusion distance [16], compromising regional blood flow [15], and increasing myocardial stiffness [17]. Reduction of interstitial edema by hyperosmotic reperfusion after ischemia has been shown to reduce infarct size [18] and improve postischemic cardiac function [19].
Recently, myocardial protection by high-dose esmolol has been introduced as an alternative to cardioplegic arrest during CABG [20]. Clinical results indicate beneficial effects of ß-blockade in patients with compromised LV function and in patients undergoing emergency CABG for acute myocardial ischemia [20, 21]. The experimental design of the presented study does not resemble the clinical situation of emergency CABG during a progressing myocardial infarction. However, the results of our study contribute further experimental evidence for the cardioprotective properties of ß-blockade as intraoperative myocardial protection.
ß-Blockade may be cardioprotective during reperfusion through various mechanisms. Infarct size after ischemia reperfusion was significantly reduced, although ß-blockade was limited to the reperfusion period. In the presence of circulatory support, high-dose ß-blockade may effectively enhance myocardial salvage, even when treatment is initiated as late as with the onset of reperfusion.
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Acknowledgments
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Supported by National Heart Lung and Blood Institute grant NHLBI-36115.
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Abstract
Introduction
