Ann Thorac Surg 1996;62:744-749
© 1996 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Effect of Cardioplegic Arrest and Reperfusion on Coronary Reserve and Autoregulation
Hidenori Gohra, MD,
Hidetoshi Tsuboi, MD,
George Sasaki, MD,
Yoshihide Minami, MD,
Kimikazu Hamano, MD,
Tomoe Katoh, MD,
Yoshihiko Fujimura, MD,
Kensuke Esato, MD
First Department of Surgery, Yamaguchi University School of Medicine, Yamaguchi, Japan
Accepted for publication April 22, 1996.
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Abstract
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Background. The effects of cardioplegic arrest and reperfusion on the coronary vasculature remain to be characterized. This study was designed to investigate changes in coronary reserve and autoregulation after hypothermic cardioplegic arrest and reperfusion.
Methods.Isolated rabbit hearts were perfused in a retrograde manner with Krebs-Henseleit bicarbonate buffer solution at a pressure of 80 cm H2O. Baseline measurements were performed for (1) coronary flow; (2) vasodilatory response to 5-hydroxytryptamine (10-7 mol/L) and nitroglycerin (10-4 mol/L); (3) autoregulatory capacity, quantified as closed-loop gains; and (4) isovolemic left ventricular function. Hearts were then subjected to cardioplegic arrest for 90 minutes. Twenty minutes after reperfusion, measurements were repeated.
Results. Coronary flow decreased significantly after reperfusion (6.2 ± 1.1 versus 5.3 ± 1.1 mL•min-1•g-1; p < 0.01). The response to 5-hydroxytryptamine as percentage increase of flow decreased significantly after reperfusion (134.0% ± 12.0% versus 109.1% ± 6.8%; p < 0.01). However, there was no significant change in the response to nitroglycerin after reperfusion (121.3% ± 17.6% versus 136.6% ± 13.3%). The closed-loop gain demonstrated negative values before arrest but became positive after reperfusion, indicating loss of autoregulation after reperfusion. There was no significant change in left ventricular function.
Conclusions.The coronary flow reserve in response to 5-hydroxytryptamine and autoregulation were impaired after cardioplegic arrest and reperfusion, whereas nitroglycerin-induced vasodilatory response and left ventricular function were preserved.
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Introduction
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Cardioplegia is the most widely used technique for myocardial protection during heart operations. Its effectiveness in protecting the myocyte has been well established in both experimental and clinical studies [1–3]. Recently, the effects of cardioplegic arrest with or without reperfusion on the coronary vascular tissue have received more attention. The coronary vasculature plays a central role in the regulation of myocardial perfusion, which, in turn, may affect myocardial dysfunction. However, alterations in coronary reserve and autoregulatory capacity after reperfusion following cardioplegic arrest have yet to be characterized. In the present study, we examined coronary flow, responses to 5-hydroxytryptamine (5-HT) and nitroglycerin (NTG) administration, autoregulatory capacity, and left ventricular function before and after cardioplegic arrest followed by reperfusion in the isolated rabbit heart.
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Material and Methods
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Surgical Procedure
Japanese white rabbits weighing 2.5 to 3.2 kg were anesthetized with 0.7 mL/kg ketamine intramuscularly and 10 mg/kg sodium pentobarbital administered through the marginal ear vein. The lungs were ventilated using a volume-controlled ventilator with 100% oxygen. Anesthesia was maintained with 1% to 2% halothane. The thorax was opened and 10 mg of heparin was given intravenously. After 30 seconds, the heart was excised and immersed in ice-cold saline solution. The aorta was cannulated, a venting tube was placed through the apex, and a latex balloon mounted on a fluid-filled catheter was positioned in the left ventricle through the mitral annulus to measure intracavitary pressure. The heart was perfused in a retrograde fashion with modified Krebs-Henseleit bicarbonate buffer solution consisting of (in mmol/L): 119.0 NaCl, 25.0 NaHCO3, 4.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2, and 11.0 glucose (pH 7.4; 37°C) at a pressure of 80 cm H2O. The buffer was equilibrated with 95% oxygen and 5% carbon dioxide.
All rabbits received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).
Dose-Response Study
To select doses of 5-HT (Sigma Chemical Co., St. Louis, MO) and NTG (The Green Cross Co, Tokyo, Japan) for the main study, we conducted a preliminary study to test the effects of 5-HT (10-9, 10-8, 10-7, 10-6, and 10-5 mol/L) and NTG (10-7, 10-6, 10-5, 10-4, and 10-3 mol/L) on coronary flow. Hearts were perfused with different concentrations of 5-HT (n = 3) or NTG (n = 3) dissolved in the buffer for 3 minutes, and coronary flow was recorded during the last 2 minutes. Between administrations of different concentrations of a drug, the coronary circulation was washed out with perfusate, and reestablishment of the basal coronary flow was confirmed before infusion of the next concentration of a drug. Based on the results of this preliminary study (Fig 1
), a 5-HT concentration of 10-7 mol/L and an NTG concentration of 10-4 mol/L were selected for further study.
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Fig 1. . Dose-response relation of coronary flow for 5-hydroxytryptamine (5-HT) (A) and nitroglycerin (NTG) (B).
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Experimental Protocol
We used 6 hearts for the main study. After 20 minutes of control perfusion with oxygenated Krebs-Henseleit bicarbonate buffer solution, basal coronary flow was measured for 2 minutes. Hearts were perfused with the buffer containing 10-7 mol/L 5-HT for 3 minutes, and coronary flow was measured during the last 2 minutes. After approximately 15 minutes of washout, during which coronary flow returned to the baseline values, the hearts were perfused with the buffer containing 10-4 mol/L NTG for 3 minutes, and coronary flow was measured during the last 2 minutes. This was followed by approximately 10 minutes of washout, during which coronary flow returned to the baseline values.
Subsequently, autoregulatory capacity was assessed by measuring steady-state coronary flow after step changes in perfusion pressure of 10 cm H2O over the range from 40 to 80 cm H2O. The closed-loop gains for the autoregulatory curves were calculated according to the following formula: G = (
F/F ÷ P/P) - 1, where G = gain, F = coronary flow, P = perfusion pressure, and F and P = the difference between the final and the initial flow and pressure, respectively. Negative values for the gain indicate autoregulation, whereas positive values indicate a dominance of passive elasticity over autoregulation [4, 5].
Then, baseline measurements of isovolemic left ventricular function (left ventricular end-diastolic pressure, left ventricular developed pressure, the positive first derivative of left ventricular pressure [dP/dt], and heart rate) were obtained at 0.1-mL increments in intraventricular balloon volume from 0 to 0.5 mL. The balloon volume was defined as 0 mL when the left ventricular end-diastolic pressure was 0 mm Hg, and the perfusion pressure was maintained at 80 cm H2O during the measurements of left ventricular function.
After these baseline measurements, the hearts were subjected to cardioplegic arrest for 90 minutes at room temperature (about 22°C). During the cardioplegic arrest, 10 mL/kg body weight of cold (4°C) hyperkalemic crystalloid cardioplegia was administered through the aortic cannula every 30 minutes at a perfusion pressure of 80 cm H2O. Cardioplegia consisted of the following: Na, 85.3 mmol/L; K, 25.0 mmol/L; Cl, 85.5 mmol/L; Mg, 10.0 mmol/L; and glucose, 25 g/L; pH was 7.38 (37°C) and osmolarity was 360 mOsm/L. Finally, the hearts were reperfused with Krebs-Henseleit bicarbonate buffer solution (37°C) at a pressure of 80 cm H2O for 20 minutes, and postreperfusion measurements of coronary flow, responses to 5-HT and NTG, autoregulatory capacity, and left ventricular function were obtained in the same manner as for the baseline measurements.
Expression of the Results
Coronary flow was measured by timed collection of the coronary effluent with the left ventricular end-diastolic pressure maintained at 0 mm Hg and the perfusion pressure maintained at 80 cm H2O, and was expressed as mL/min per gram wet heart weight. Measurements used for determining the response of coronary flow to 5-HT or NTG were obtained during the last 2 minutes of drug-free perfusion before changing to drug-containing buffer (control) and during the last 2 minutes of perfusion with drug-containing buffer. The responses to 5-HT or NTG in coronary flow were expressed as a percentage of the control value: Percentage increase (%) = respondent coronary flow/control coronary flow x 100.
All data in the text, table, and figures are expressed as mean ± standard deviation. Statistical analysis was performed using the paired t test. Values were considered significantly different at p less than 0.05.
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Results
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Dose-Response Study
Figure 1
shows the dose-response relation of coronary flow to the doses of 5-HT at 10-9, 10-8, 10-7, 10-6, and 10-5 mol/L and the doses of NTG at 10-7, 10-6, 10-5, 10-4, and 10-3 mol/L. A 5-HT dose of 10-7 mol/L and an NTG dose of 10-4 mol/L were chosen for the following experiments because these doses induced threshold or maximal responses without significant changes in heart rate or contractile function.
Coronary Flow
The baseline coronary flow was 6.2 ± 1.1 mL • min-1• g-1. Postreperfusion coronary flow decreased significantly to 5.3 ± 1.1 mL•min-1•g-1 after reperfusion following cardioplegic arrest (p < 0.01; Fig 2).
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Fig 2. . Coronary flow before cardioplegic arrest and after reperfusion. The coronary flow decreased significantly after reperfusion following cardioplegic arrest.
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Responses to 5-Hydroxytryptamine and Nitroglycerin Administration
There was a significant increase in coronary flow in response to 5-HT from baseline before cardioplegic arrest (6.2 ± 1.1 versus 8.2 ± 1.2 mL•min-1•g-1; p < 0.01), which could not be demonstrated after reperfusion (5.2 ± 1.0 versus 5.7 ± 1.2 mL•min-1•g-1) (Fig 3). The percentage increase over control was significantly lower after reperfusion compared with before cardioplegic arrest (134.0% ± 12.0% versus 109.1% ± 6.8%; p < 0.01) (Fig 4). The coronary flow in response to NTG showed a significant increase from control both before cardioplegic arrest (6.1 ± 1.0 versus 8.4 ± 1.1 mL•min-1•g-1; p < 0.01) and after reperfusion (5.3 ± 1.1 versus 7.3 ± 1.1 mL • min-1•g-1; p < 0.01) (see Fig 3), and there was no significant difference in the percentage increase before cardioplegic arrest versus after reperfusion (121.3% ± 17.6% versus 136.6% ± 13.3%) (see Fig 4).
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Fig 3. . Coronary flow (CF) in response to 5-hydroxytryptamine (5-HT) or nitroglycerin (NTG) before cardioplegic arrest (Pre.) and after reperfusion (Post.). 5-Hydroxytryptamine–induced coronary flow increased significantly compared with control coronary flow before cardioplegic arrest, but not after reperfusion. Nitroglycerin-induced coronary flow increased significantly compared with control coronary flow both before arrest and after reperfusion.
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Fig 4. . Percentage increase in coronary flow induced by 5-hydroxytryptamine (5-HT) and nitroglycerin (NTG) before cardioplegic arrest and after reperfusion. The percentage increase in response to 5-hydroxytryptamine showed a significantly lower value after reperfusion than before cardioplegic arrest. However, there was no significant difference in the percentage increase induced by nitroglycerin before cardioplegic arrest and after reperfusion.
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Autoregulatory Capacity
To quantify the pressure-flow relation under steady-state conditions, we altered the coronary perfusion pressure between 40 and 80 cm H2O; the calculated gains are shown in Figure 5. The gains were found to be negative values at perfusion pressures between 50 and 80 cm H2O before cardioplegic arrest. In contrast, after reperfusion the gains became positive, with the exception of perfusion pressure between 70 and 80 cm H2O.
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Fig 5. . Pressure–gain relations. Autoregulation was maintained at a perfusion pressure between 50 and 80 cm H2O before cardioplegic arrest, but only in a narrow range of perfusion pressure between 70 and 80 cm H2O after reperfusion.
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Isovolemic Left Ventricular Function
Left ventricular function (left ventricular end-diastolic and developed pressures, dP/dt, and heart rate) was essentially unchanged after reperfusion following cardioplegic arrest (Table 1).
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Comment
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The present study was undertaken to examine the alterations in coronary flow, coronary reserve, autoregulatory capacity, and left ventricular function after cardioplegic arrest and subsequent reperfusion. The major findings of the present study were as follows: (1) Nonstimulated coronary flow decreased after cardioplegic arrest and reperfusion; (2) vasodilation of the coronary artery in response to 5-HT is impaired after reperfusion, whereas NTG-induced vasodilation is unaffected by cardioplegic arrest and reperfusion; (3) autoregulation of coronary flow in response to changes in perfusion pressure is impaired after reperfusion; and (4) left ventricular function is preserved after cardioplegic arrest and reperfusion.
Endothelial cells play an important role in the regulation of coronary artery tone through the release of endothelium-derived relaxing factor [6–9], which has been identified as nitric oxide [10]. Studies have shown that 5-HT causes vasodilation by stimulating the release of endothelium-derived relaxing factor (ie, nitric oxide) [11–13], and has been used to assess endothelial function [14–16]]. Our results demonstrated that coronary flow reserve in response to 5-HT was impaired after reperfusion, which suggests that endothelial dysfunction may be caused by cardioplegic arrest or reperfusion. Endothelial dysfunction could reduce the basal release of endothelium-derived relaxing factor, resulting in the decrease in coronary flow after reperfusion shown in Figure 2. In addition, endothelial dysfunction could lead to an increase in permeability of the coronary vasculature, with ensuing interstitial and intracellular edema. This edema could cause extravascular compression of the coronary arteries and contribute to the decrease in coronary flow after reperfusion. Previous studies have shown that crystalloid cardioplegic arrest with or without reperfusion causes impairment of endothelium-derived relaxing factor release [14–17] as well as structural damage to endothelial cells [18, 19]. Which is responsible for endothelial damage-cardioplegia per se, reperfusion, or both-remains controversial. Saldanha and Hearse [14] have shown that infusion of hyperkalemic crystalloid cardioplegic solution, without ischemia, causes endothelial damage. Potassium ions or hypothermia might induce endothelial dysfunction. On the other hand, Evora and associates [20] demonstrated that hyperkalemic crystalloid cardioplegia does not alter function of the epicardial coronary artery. Cartier and associates [21] studied the effects of perfusion pressure of hyperkalemic crystalloid cardioplegic solutions and demonstrated that an infusion pressure of 80 cm H2O does not cause impairment of 5-HT–induced vasodilation. Free radicals produced by ischemia/reperfusion could cause endothelial dysfunction [22].
Nitroglycerin induces dilation of coronary arteries by acting on the vascular smooth muscle, and its action is independent of endothelial function. Our results demonstrated no significant difference in the percentage increase in coronary flow in response to NTG before cardioplegic arrest versus after reperfusion, indicating that the coronary flow reserve in response to NTG was preserved. The finding that 5-HT–induced vasodilatory response was impaired whereas NTG-induced vasodilation was maintained after reperfusion suggests that the coronary vascular endothelium is more vulnerable to cardioplegic arrest and reperfusion than is vascular smooth muscle.
Coronary autoregulation is defined as the intrinsic ability of the coronary circulation to maintain a relatively constant blood flow supply in response to changes in perfusion pressure over a wide range [23]. In the present study, we examined the steady-state pressure-flow relation of the coronary artery and quantified the degree of autoregulation by measuring the autoregulatory gain [4, 5]. The gain demonstrated negative values in a range of perfusion pressure between 50 and 80 cm H2O before cardioplegic arrest, but only in a narrow range between 70 and 80 cm H2O after reperfusion. This indicates that the autoregulatory capacity of the coronary artery was abolished after reperfusion. It has been postulated that coronary autoregulation may be linked to myocardial metabolism, ie, oxygen consumption [23, 24]. Although we did not measure oxygen consumption in the present study, hearts were perfused in a nonworking mode, and few differences were found in left ventricular developed pressure and heart rate after reperfusion. This suggests that the myocardial oxygen consumption is not significantly altered after reperfusion. Endothelial dysfunction could contribute, in some part, to the abolishment of coronary autoregulation through impairment of endothelium-dependent vasodilatory reserve and through the development of vascular, myocardial, and interstitial edema.
Although there was a trend toward increased left ventricular end-diastolic pressure and lower developed pressure and positive dP/dt after reperfusion following cardioplegic arrest, there was little statistical difference in isovolemic left ventricular function before cardioplegic arrest versus after reperfusion. Taken together with the findings of decreased basal coronary flow and responsiveness to endothelium-dependent vasodilator stimuli, as well as the abolished coronary autoregulation, the coronary vasculature appears be more vulnerable to cardioplegic arrest and to reperfusion than is the myocyte. Our results also suggest that the impairment of endothelial function might have little effect on left ventricular function.
In summary, we examined the alterations of coronary flow, coronary reserve, autoregulation, and left ventricular function of the isolated rabbit heart subjected to 90 minutes of cardioplegic arrest induced with a hypothermic crystalloid cardioplegic solution and reperfusion. Coronary flow decreased significantly and endothelium-dependent coronary vasodilatory reserve and coronary autoregulation were impaired, whereas endothelium-independent coronary vasodilatory reserve and myocardial function were preserved.
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Footnotes
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Address reprint requests to Dr Gohra, First Department of Surgery, Yamaguchi University School of Medicine, 1144 Kogushi, Ube, Yamaguchi 755, Japan.
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References
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Abstract
Introduction
