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CARDIOVASCULAR ANESTHESIA

Aprotinin Pretreatment Diminishes Postischemic Myocardial Contractile Dysfunction in Dogs

Department of Anesthesiology, Rush Medical College at Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois

Address correspondence and reprint requests to Kenneth J. Tuman, MD, Rush Medical College at Rush-Presbyterian-St. Luke’s Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612.

	Abstract

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We evaluated the effect of aprotinin, administered before the onset of acute regional myocardial ischemia, on reversible contractile dysfunction induced by ischemia and reperfusion in pentobarbital-anesthetized dogs. Animals were randomized to receive either aprotinin 30,000 kallikrein inactivator units (KIU)/kg and 7000 KIU · kg^-1 · hr^-1 (n = 8) or equivalent volumes of 0.9% sodium chloride (n = 7) IV 60 min before a 15-min interruption of circumflex coronary artery blood flow and then reperfusion. There were no intra- or intergroup differences in hemodynamic variables or regional myocardial mechanics (sonomicrometry) before onset of ischemia. Immediately before reperfusion, systolic dysfunction characterized by significantly decreased percent systolic shortening was present in the circumflex coronary artery area of both study groups. After reestablishment of perfusion, aprotinin animals had preserved percent systolic shortening whereas saline animals exhibited regional systolic dysfunction. Regional myocardial contractility as assessed by the slope M_w of the preload recruitable stroke work relation was preserved during reperfusion in animals who received aprotinin but depressed in the control group. We conclude that functional recovery from myocardial ischemia-reperfusion injury at normothermia is improved by IV administration of aprotinin before the onset of acute regional myocardial ischemia in physiologically intact dogs.

Implications: Administration of clinically relevant doses of aprotinin IV before the onset of regional myocardial ischemia, in contrast to control conditions, preserved regional systolic function and contractility at baseline values after reestablishment of myocardial perfusion in dogs.

	Introduction

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Ischemia-reperfusion injury involves inflammatory processes mediated by vascular endothelium, white blood cells, and platelets (1). Although used during cardiac surgical procedures to decrease bleeding and transfusion, the antiinflammatory actions of aprotinin might convey a beneficial effect on the pathophysiologic events of myocardial ischemia-reperfusion injury. Large-dose aprotinin has been associated with reduced cardiac troponin T levels after cardiopulmonary bypass, suggesting a reduction in ischemic myocardial damage after reperfusion (2). Clinical reports also have suggested that aprotinin can reduce the extent of myocardial injury (creatine kinase release, ST segment changes, and Q wave evolution) when administered to patients soon after the onset of acute myocardial ischemia (3).

Previous animal investigations of the effects of aprotinin on myocardial function after ischemia reperfusion present conflicting results. Aprotinin attenuates the decrements in load-dependent measures of contractility observed after global ischemia and reperfusion in isolated rat hearts (4), whereas isolated canine hearts perfused with cold cardioplegia containing aprotinin exhibit more impaired contractility than after cardioplegia without aprotinin (5). In models of more severe acute ischemic injury, aprotinin either diminishes or increases myocardial necrosis after acute coronary occlusion (6,7). Animal data also suggest that aprotinin can attenuate the protective effect of ischemic preconditioning (7). However, no data are available to evaluate the in vivo effects of aprotinin on myocardial function during reperfusion after less severe, reversible regional myocardial ischemia (stunning).

We designed this study to evaluate the effects of aprotinin, administered before the onset of acute regional myocardial ischemia, on the reversible contractile dysfunction induced by ischemia and reperfusion in physiologically intact animals.

	Methods

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After approval of the institutional animal care and use committee, 15 mongrel dogs (27 ± 3.5 kg) of either sex were fasted overnight and then anesthetized with sodium pentobarbital 30 mg/kg before an infusion of 1 mg · kg^-1 · h^-1. After tracheal intubation, positive pressure ventilation was used to maintain arterial PCO₂ between 35 to 42 mm Hg and PO₂ between 150 to 200 mm Hg by adjusting respiratory rate, tidal volume, and inspired oxygen concentration. Body temperature was monitored continuously and normothermia was maintained with a surface-heating blanket and warmed IV fluids.

A pressure transducer-tipped catheter was inserted via the carotid artery to measure aortic blood pressure at the level of the aortic valve. The ipsilateral jugular vein was cannulated for infusion of 0.9% sodium chloride solution at 4 mL · kg^-1 · h^-1 throughout the experiment. A lateral thoracotomy was performed, the pericardial sac opened, and the heart suspended in a pericardial cradle. A pressure transducer-tipped catheter was inserted through the left ventricular (LV) apex for measurement of LV pressures and the rate of change of LV pressure (dP/dt) by analog electronic differentiation. Pressure transducers (SPC 450; Millar Instruments, Houston, TX) were calibrated before insertion in 0.9% sodium chloride at 39°C; after insertion and during the experiment, pressure transducers were adjusted for zero drift against a fluid-filled catheter placed in the left atrium. Appropriately sized electromagnetic flow probes (Flo-Probe^TM Bloodflow Tranducers; Gould, Inc., Oxnard, CA) were placed around the proximal ascending aorta and the circumflex coronary artery (CIRC) distal to its first marginal branch. The flow probes were set at the factory calibration and zeroed in 0.9% sodium chloride before placement. After placement, zero adjustment was performed electronically (SP2202 Bloodflow Transducer; Gould, Inc.). At the end of the experiment, calibration and offset adjustments were determined by timed volume collection. A mechanical snare (0 silk ligature) was loosely placed around the circumflex artery distal to the flowprobe for subsequent flow interruption. No testing of the snare was undertaken before total flow interruption to avoid any preconditioning effects. We considered complete vessel occlusion to have been achieved when there was absence of pulsatile flow detected by the distal flowprobe. Two pairs of cylindrical ultrasonic segment length transducers (5 MHz) were implanted to a depth of 8 mm in the myocardial perfusion territories of the CIRC and left anterior descending coronary artery (LAD) for measurement of regional contractile function. Ultrasonic amplifiers (Triton Technologies, San Diego, CA) were used to calibrate and monitor segment length signals.

Data collection was performed by analog to digital conversion at 200 Hz with 16-bit resolution using the Cordat II^TM (Data Integrated Scientific Systems, Pickney, MI) software system. The time constant of isovolumic relaxation () was calculated assuming a nonzero asymptote of the LV pressure decay from the maximum negative dP/dt to the pressure at mitral valve opening estimated at end diastolic pressure plus 5 mm Hg (8). End systolic myocardial segment length (ESL) was measured at 10 ms before maximum –dP/dt and end diastolic myocardial segment length (EDL) was measured immediately before the onset of LV isovolumic contraction. Regional wall motion (% systolic shortening, %SS) was quantified using the formula (%SS) = (EDL – ESL)/EDL x 100.

The animals were randomized to receive either aprotinin 30,000 kallikrein inactivator units (KIU)/kg and 7000 KIU · kg^-1 · h^-1 (n = 8) or equivalent volumes of 0.9% sodium chloride (n = 7) IV. Sixty minutes after study drug bolus, hemodynamic and myocardial segment measurements were recorded before and at the end of a 15-min interruption of the CIRC blood flow and subsequently (15, 30, 45, and 60 min) after the onset of reperfusion. In addition, before the onset of ischemia, as well as 15, 30, and 60 min after the onset of reperfusion, the inferior vena cava was constricted briefly to obtain pressure-segment length data necessary for determination of the slope (M_w) and intercept (L_w) of the preload recruitable stroke work relationship as described previously (9,10).

Time-averaged values determined over a 1-min epoch were used to describe hemodynamic and regional wall motion variables at the reported intervals. Pressure-segment length data used to determine M_w and L_w were derived from a minimum of 10 cardiac cycles. Data were compared within and between groups over time with multivariate analysis of variance for repeated measures. Post hoc testing was performed using the Tukey method at = 0.05 when analysis of variance identified differences with P < 0.05. Data are expressed as mean ± SEM.

	Results

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Before the administration of study drug, and 60 min after administration of study drug (preischemic baseline), there were no intra- or intergroup differences in mean aortic pressure, heart rate, left ventricular stroke volume, left ventricular end diastolic pressure, LV dP/dt, diastolic CIRC blood flow, and (Table 1). There were no differences in preischemic baseline values in the CIRC perfusion area of EDL (13.2 ± 0.7 vs 15.1 ± 1.4 mm) or of ESL (12.0 ± 0.7 vs 14.1 ± 1.1 mm) among the saline and aprotinin groups, respectively. Similarly, preischemic baseline values of EDL (14.9 ± 1.1 vs 12.4 ± 0.9 mm) and ESL (12.7 ± 0.9 vs 10.7 ± 0.9 mm) in the LAD perfusion area did not differ among groups. Calculated values of %SS did not differ among groups before ischemia (Figure 1: CIRC perfusion area and Figure 2: LAD perfusion area). Likewise, M_w and L_w did not differ among or within groups in either myocardial perfusion area before the onset of ischemia (Table 1).

View larger version (24K): [in this window] [in a new window]   Figure 1. Percent systolic segment shortening in the circumflex coronary artery (CIRC) perfusion area at preischemic baseline, during 15 min of CIRC flow interruption and during reperfusion in dogs that received aprotinin (n = 8) versus saline (n = 7). Values are mean ± SEM. = Significantly different from aprotinin, = 0.05, = Significantly different from pre-ischemic baseline within group, = 0.05.

View larger version (24K): [in this window] [in a new window]   Figure 2. Percent systolic segment shortening in the left anterior descending coronary artery (LAD) perfusion area at preischemic baseline, during 15 min circumflex coronary artery flow interruption and during reperfusion in dogs that received aprotinin (n = 8) versus saline (n = 7). Values are mean ± SEM. No values significantly different within groups at any time compared with preischemic baseline or among groups at any time.

After reestablishment of blood flow to the CIRC perfusion area, there were no intergroup differences in global hemodynamic variables (Table 1). No differences in %SS compared with preischemic baseline were observed in the aprotinin group (Figure 1). In the saline group, CIRC %SS was decreased from preischemic baseline as well as compared with the aprotinin group after reestablishment of CIRC blood flow. There were no differences in %SS among or within groups in the nonischemic LAD perfusion area (Figure 2).

Regional contractility in the CIRC perfusion area, as assessed by the slope M_w of the preload recruitable stroke work relation, was reduced at 15 and 30 min of reperfusion in the saline group but at none of the data collection times in the aprotinin group (Table 1). There were no changes in the intercept (L_w) of the preload recruitable stroke work relation during reperfusion in either group.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major findings of this investigation were important differences among the study groups in regional systolic myocardial performance during reperfusion after ischemia. In contrast with control conditions, administration of clinically relevant doses of aprotinin before the onset of ischemia preserved systolic function (%SS) at baseline values after reestablishment of perfusion. In addition, regional contractility (reflected by M_w) was preserved during reperfusion in animals that received aprotinin, whereas it was depressed in the control group.

These findings may differ from previously published data because the experimental model of myocardial stunning applied in our study permitted examination of regional myocardial mechanics in the physiologically intact animal (rather than in isolation) (4,5) and because the severity of ischemia was less than in previous studies in which the major endpoint was the amount of myocardial necrosis (6,7).

Myocardial ischemia reperfusion is accompanied by inflammatory processes mediated by vascular endothelium, neutrophils, and platelets. Cytokines, such as tumor necrosis factor- (TNF-) and interleukins, result in margination and adherence of inflammatory cells to the endothelial surface. Cytokines such as TNF- cause the prompt (two to four minutes) expression of neutrophil integrins such as CD11b, which mediate neutrophil-mediated myocardial reperfusion injury (11,12). Interleukin-6 (IL-6) mRNA synthesis is accelerated rapidly by reperfusion of ischemic myocardium and immediately precedes synthesis of intercellular adhesion molecule-1 which binds to neutrophil adhesion molecules such as CD11b, resulting in neutrophil attachment to endothelium and subsequent transendothelial migration of neutrophils (13). IL-6 (and to a lesser extent IL-8) mediates the myocardial dysfunction observed as a manifestation of ischemia-reperfusion injury (14). Stimulated leukocytes can secrete a number of cytotoxic moieties, including oxygen free radicals and proteolytic enzymes. In addition, endogenous nitric oxide concentrations are increased during myocardial reperfusion injury (15).

Although the mechanism of aprotinin’s beneficial effects on myocardial stunning is unclear, an antiinflammatory role involving several of the aforementioned processes can be postulated based on several lines of evidence. Serine protease inhibition suppresses complement activation in response to myocardial ischemia, so that important mediators such as C5a do not produce neutrophil activation and endothelial injury (16). In vitro data indicate that aprotinin can inhibit the production and release of free radicals from activated neutrophils (17,18). Likewise, aprotinin inhibits cytokine-induced nitric oxide synthase expression in vitro (19), limiting the increases in endogenous cytokine-induced nitric oxide production which have been causally associated with myocardial reperfusion injury (15). In the setting of cardiopulmonary bypass (CPB), aprotinin attenuates the increases in blood levels of TNF-, blunting neutrophil integrin CD11B up-regulation (20). IL-6 release during CPB is also inhibited by aprotinin (21). Because increasing levels of IL-6 correlate with myocardial reperfusion injury (14), therapeutic interventions that reduce IL-6 concentrations have been postulated to reduce the severity of myocardial reperfusion injury (21). Our findings are consistent with that hypothesis. Although aprotinin modulates the vasomotor responses to a thromboxane analog in coronary bypass grafts (22), we did not observe any differences in coronary blood flow between aprotinin and placebo animals at any of the data measurement times during this study.

Our study did not attempt to elucidate the mechanism of the protective effect of aprotinin, or determine whether the attenuation of functional impairment after myocardial stunning was dose dependent. Although aprotinin levels were not measured, the aprotinin dose we used should produce blood and tissue concentrations approximating those achieved after doses that are often given to humans for CPB. We acknowledge that a limitation of the study is the absence of control for myocardial collateral blood flow. It is possible that there were intergroup differences in collateral blood flow that could in part account for some of the findings. It is unknown whether any such putative differences in collateral blood flow might be directly or indirectly related to acute effects of aprotinin. Because the experiment was not designed to define the precise mechanism of aprotinin’s beneficial actions on preserving postischemic contractile function, further investigations will be required to evaluate such a potential mechanism.

Species differences might contribute to differing effects of aprotinin pretreatment on myocardial stunning, possibly because of differences in coronary collateral circulation. Although more intense transmural myocardial ischemia occurs after coronary artery occlusion in species with less developed coronary collaterals (e.g., pigs and baboons), more severe myocardial stunning is observed after a single flow interruption (of similar duration to our study) in dogs than in species with less developed coronary collaterals (23). Another limitation of this study was the evaluation of aprotinin’s effects on ischemia and reperfusion in a model using normal myocardium and coronary vessels. This factor precludes the extension of the findings to the scenario in which aprotinin would be administered before onset of stunning of myocardium burdened with preexisting ischemia related to coronary artery disease. Conversely, our model eliminates the confounding effects of hypothermia on the results of an intervention to attenuate ischemia-reperfusion injury, and may explain the differences in our findings compared with other work showing that isolated canine hearts, perfused with cold cardioplegia containing aprotinin, had more impaired contractility than after cardioplegia without aprotinin (5).

In summary, our study shows that functional recovery from myocardial ischemia-reperfusion injury at normothermia is improved by IV administration of aprotinin before the onset of acute regional myocardial ischemia in physiologically intact dogs.

	References

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999