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Ann Thorac Surg 1997;63:41-49
© 1997 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Effects of Blood and Crystalloid Cardioplegia on Adrenergic and Myogenic Vascular Mechanisms

Division of Cardiothoracic Surgery, Department of Surgery, Beth Israel Hospital, and Harvard Medical chool, Boston, Massachusetts

Accepted for publication July 5, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Conclusion Acknowledgments References

 
Background. This study compares the effects of cold blood and crystalloid cardioplegia on adrenergic and myogenic regulation of the coronary circulation.

Methods. Pigs were placed on cardiopulmonary bypass and hearts were arrested with a hyperkalemic crystalloid cardioplegic solution (Cryst CP) or blood cardioplegic solution (Blood CP) for 1 hour. Hearts of selected pigs were then reperfused for 1 hour (Rep) and separated from cardiopulmonary bypass. Left ventricular perfusion and contractility and ß- and ₂-adrenergic and myogenic responses of the coronary circulation were examined.

Results. Relaxation of isolated, precontracted microvessels to isoproterenol (ß-adrenoceptor agonist) was reduced to a lesser extent after Blood CP as compared with Cryst CP. Relaxation to forskolin (adenylate cyclase activator) was reduced after Cryst CP, but was preserved after Blood CP. After 1 hour of postcardioplegia reperfusion, the respective responses to isoproterenol and forskolin were similar in vessels from the Cryst CP-Rep and Blood CP-Rep groups. The ₂-adrenoceptor-mediated, endothelium-dependent vascular relaxation to clonidine was decreased more after Cryst CP than after Blood CP. The relaxation to nitroprusside was not affected by either Cryst CP or Blood CP. Myogenic tone was decreased to a lesser extent after Blood CP versus Cryst CP. Baseline coronary blood flow, isoproterenol-induced increases of coronary blood flow, and indices of myocardial contractility were similar in the Blood CP-Rep and Cryst CP-Rep groups, both 5 and 60 minutes after initiation of reperfusion.

Conclusions. Although Blood CP was superior to Cryst CP in preserving ß- and ₂-adrenoceptor function and myogenic tone in vitro, there was no demonstrable benefit of blood cardioplegia in the preservation of myocardial contractility or perfusion in this model of cardioplegia.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Conclusion Acknowledgments References

 
Episodic coronary spasm has been reported to occur in approximately 2.5% of patients after cardiac operation [1] and may have devastating effects on the outcome of a cardiac operation. Postoperative coronary artery contraction may be the result of endothelial dysfunction and changes in vascular smooth muscle reactivity. Contributing to this episodic increased coronary vascular tone are the increased circulating and local tissue levels of catecholamines [2, 3], thromboxane [4] and other products of platelet activation, and other constrictor substances during and after cardiopulmonary bypass (CPB) and cardioplegia. The consequence of cardioplegia on vascular reactivity has been the subject of numerous experimental studies [5–9]. In general, endothelium-dependent responses are impaired after crystalloid cardioplegia [6], although the impairment may not be as pronounced in large epicardial conduit arteries [7] as that observed in the coronary microcirculation [6, 8]. The addition of blood to a cardioplegic solution lessens the reduction in this aspect of endothelial function [8, 10]. Although having received much less attention than endothelium-dependent mechanisms, direct vascular smooth muscle pathways of vasomotor regulation are also important determinants of coronary vascular tone. ß-Adrenergic receptor stimulation causes potent relaxation in the coronary circulation through a cyclic adenosine monophosphate-mediated mechanism [11], whereas stimulation of ₂-adrenoceptors induces endothelium-dependent relaxation in the coronary circulation [11, 12]. Therefore, preservation of ß- and ₂-adrenergic mechanisms in coronary circulation may improve myocardial perfusion after cardioplegia and thereby may improve functional recovery of the heart.

Myogenic tone is a property of vascular smooth muscle manifested by contraction in response to increases in transmural pressure, and is involved in the autoregulation of organ perfusion. Although not completely understood, myogenic contraction is thought to be mediated largely through the activation of protein kinase C, is dependent on extracellular calcium, and independent of an endothelial influence [13, 14]. We have found that myogenic tone of coronary microvessels is decreased after cardiopulmonary bypass and crystalloid cardioplegia [14]. Changes in myogenic tone may affect both coronary and peripheral vascular tone and therefore, resistance to blood flow.

The goal of this study was to determine the possible differential effects of cold blood and crystalloid hyperkalemic cardioplegia on the preservation of coronary microvascular ß- and ₂-adrenoceptor-mediated responses and myogenic contraction under conditions of CPB, and to correlate the effects of cardioplegia on vascular responses to coronary blood flow (CBF) and myocardial contractility.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Conclusion Acknowledgments References

 
Animal Preparation
Yorkshire pigs (20 to 25 kg) of either sex were premedicated with intramuscular ketamine (10 mg/kg) and anesthetized with intravenous alpha-chloralose and urethane (60 mg/kg and 300 mg/kg initially and 15 mg/kg and 60 mg/kg every 30 to 60 minutes as needed, respectively). Pigs were tracheally intubated and mechanically ventilated. In the control group (n = 6), a sternotomy was performed and pigs were heparinized (500 U/kg). The hearts were rapidly excised and immediately placed in cold (5° to 10°C) Krebs' buffer solution of the following composition (in mmol/L): NaCl, 118.3; KCl, 4.7; CaCl₂, 2.5; MgSO₄, 1.2; NaH₂PO₄, 1.2; NaHCO₃, 25; and glucose, 11.1. All animals received humane care in compliance with the Animal Care and Use Committee of Beth Israel Hospital and with the "Principles of Laboratory Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 86-23, revised 1985).

Cardiopulmonary Bypass Preparation
After induction of anesthesia and tracheal intubation, a fluid-filled catheter was introduced into the femoral artery and vein for vascular access. After a sternotomy was made, pigs were heparinized (500 U/kg initially and 200 U/kg 2 hours later) and cannulated through the distal ascending aorta and the right atrium. Activated clotting time was measured and maintained at more than 500 seconds. A left ventricular vent was placed through the left ventricular apex for decompression. A small cannula for the infusion of cardioplegic solution was inserted into the ascending aorta through a pursestring suture. Normothermic CPB was instituted using a bubble oxygenator (Bentley Bio-2; Baxter Healthcare Corp, Irvine, CA) and a roller pump. An arterial filter (Bentley Bio-1025; Baxter Healthcare) was inserted into the circuit distal to the roller pump. Blood flow was maintained between 2.0 and 3.0 L/min (2.5 to 4.0 L • min^-1 • m^-2) to maintain a mean perfusion pressure of between 50 and 80 mm Hg. Systemic blood temperature was maintained at 37°C. Arterial blood gases before and after CPB were maintained by ventilatory rate, tidal volume, and fractional concentration of oxygen to keep the partial pressure of oxygen at more than 100 mm Hg, pH between 7.35 and 7.45, and the partial pressure of carbon dioxide at more than 30 and less than 45 mm Hg.

Crystalloid and Blood Cardioplegia
After stabilization of the preparation for CPB, an aortic cross-clamp was placed between the aortic perfusion cannula and the cardioplegia infusion cannula. Three hundred milliliters of cold (4°C) hyperkalemic ([K⁺] = 25 mmol/L) crystalloid cardioplegic solution (n = 6) or cold (4°C) blood cardioplegic solution (hematocrit 16%, n = 6) was infused into the aortic root at an infusion pressure of 60 mm Hg. The composition of the crystalloid cardioplegic solution was (in mmol/L): NaCl, 121; KCl, 25; NaHCO₃, 12; and glucose, 11.1 (pH = 7.6 and partial pressure of oxygen range = 180 to 300 mm Hg). Blood cardioplegic solution consisted of equal volumes of crystalloid cardioplegic solution and blood removed from the pig. Potassium chloride was supplemented to raise the K⁺ concentration to 25 mmol/L. Saline slush was placed on the surface of the heart for topical hypothermia during the time the aorta was cross-clamped. Myocardial temperature in the distribution of the left anterior descending artery was measured with a probe and ranged from 6 to 14°C during the period of cardioplegic arrest. In both experimental groups crystalloid (Cryst CP) and blood (Blood CP) cardioplegia, infusion of the respective cardioplegic solution (150 mL) was repeated at 20-minute intervals for 60 minutes (two additional administrations). After a total of 60 minutes of cardioplegia, the hearts were excised and immediately placed in cold Krebs' buffer solution.

Cardioplegia–Reperfusion
The same procedure was performed as in the cardioplegia experiments. However, after 60 minutes of cardioplegic arrest, the aortic cross-clamp was removed and the heart was reperfused (Rep) for 60 minutes with normothermic blood in the bypass circuit (Cryst CP-Rep, n = 6; Blood CP-Rep, n = 6). In the event of ventricular fibrillation, the rhythm was converted to sinus with 10 J after the myocardial temperature rose to more than 30°C. The pig was separated from extracorporeal support and was then decannulated. This generally occurred within 15 minutes after cross-clamp removal. After a total of 60 minutes of reperfusion, the pig was euthanized by exsanguination under anesthesia, the heart was rapidly excised and immediately placed in cold Krebs buffer solution. In an effort to reduce the number of animals used in this study, pigs in the control (n = 2), Cryst CP (n = 2), and Cryst CP-Rep (n = 2) groups were included from those used in previous studies and reported in other articles [5, 14]. Experiments were performed on additional pigs de novo (n = 4 in each group) to obtain the numbers described.

In Vivo Effects of Isoproterenol on Coronary Blood Flow and Myocardial Function
In vivo responses to isoproterenol were studied in pigs from the Blood CP-Rep and Cryst CP-Rep groups. Pigs were instrumented before CPB. An 8F micromanometer-tipped catheter (Millar Instruments, Houston, TX) was inserted through the left ventricular apex into the left ventricular cavity. The maximum rate of increase of left ventricular pressure to the first derivative of left ventricular pressure (dP/dt) was derived by differentiating the left ventricular pressure signal over time. In preparation for the intracoronary administration of isoproterenol, a Silastic catheter (inner diameter, 0.3 mm; outer diameter, 0.5 mm) was introduced into the proximal left anterior descending artery. The CBF was measured with an ultrasonic flow probe (Transonic Systems Inc, Ithaca, NY), which was placed around the left anterior descending artery distal to the point of cannulation for intracoronary drug administration. A pair of sonomicrometer crystals was implanted in the left ventricular subendocardium perfused by the left anterior descending artery branches. The crystals were placed parallel to the short axis of the left ventricle. Percent systolic shortening was calculated as: (end-diastolic segment length - end-systolic segment length/end-diastolic segment length) x 100.

In vivo responses to intracoronary isoproterenol administration were studied before initiation of CPB, 5 minutes after discontinuation of cardioplegic arrest, and after 1 hour of postcardioplegia reperfusion. Isoproterenol was administered at a rate of 0.02 µg • kg^-1 • min^-1 for 2 minutes (volume, 0.6 mL/min) using a miniature pump (Living System Instrumentation, Burlington, VT). The recording was first taken at 20 seconds after the initiation of drug administration, while dP/dt, fractional shortening, and heart rate were unchanged. The second recording was taken after approximately 2 minutes, while hemodynamic variables and cardiac functional parameters had usually been altered due to isoproterenol-induced inotropic and chronotropic effects. Pressure and flow signals were recorded on an eight-channel recorder (Honeywell-Electronics for Medicine Research, Natick, MA).

In Vitro Coronary Microvessel Studies
Coronary arterial microvessels (internal diameter, 80 to 170 µm) were dissected from the subepicardial region of the left ventricle of each heart using a 10 to 60x dissecting microscope (Olympus, Tokyo, Japan). The left anterior descending artery-dependent myocardium proximal to the point of coronary cannulation was selected. When examining agonist-induced responses, microvessels were placed in a Plexiglass organ chamber, cannulated with dual glass micropipettes measuring 30 to 80 µm in diameter and secured with 10-0 nylon monofilament suture (Ethicon, Somerville, NJ). Oxygenated (95% O₂, 5% CO₂) Krebs' buffer solution warmed to 37°C was continuously circulated through the organ chamber and a reservoir (total fluid volume, 100 mL). Microvessels were pressurized to 40 mm Hg in a no-flow state using a burette manometer filled with Krebs' buffer solution.

In preparation for myogenic experiments, a similar procedure as described above was performed. Both ends of the micropipettes were connected to a pressure reservoir; therefore, the intraluminal pressure could be varied by adjusting the height of the reservoir. 3-(N-morpholino) Propanesulfonic acid–albumin (1 g human albumin to 100 mL) buffer solution (composition in mmol/L: NaCl, 145.0; KCl, 4.7; CaCl₂, 2.0; MgSO₄, 1.2; glucose, 5.0; pyruvate, 2.0; EDTA, 0.02; NaH₂PO₄, 1.2; and 3-(N-morpholino) propanesulfonic acid, 3.0) was continuously circulated through the organ chamber. The solution was warmed to 37°C by an external heat exchanger and equilibrated with room air.

An inverted microscope (40 to 200x; Olympus) was connected to a video camera and the vessel image was projected onto a black and white television monitor (Hitachi, Japan). An electronic dimension analyzer (Living System Instrumentation, Burlington, VT) was used to measure internal lumen diameter. Measurements were recorded with a stripchart recorder (Graphtec, Irvine, CA). Vessels were allowed to equilibrate for at least 30 minutes in Krebs' buffer solution before a drug intervention.

Evaluation of Adrenergic-, Cyclic Adenosine Monophosphate-, and Cyclic Guanosine Monophosphate-Mediated Responses
In all experimental groups, relaxations of microvessels were examined after precontraction with the thromboxane A₂ analog U46619 by 25% to 40%. Once the steady-state tone was reached, the dose responses to isoproterenol (10^-12 to 10^-4 mol/L), forskolin (10^-9 to 10^-5 mol/L), clonidine (10^-9 to 10^-4 mol/L), or sodium nitroprusside (10^-9 to 10^-4 mol/L) were examined. One to three interventions were performed on each vessel. The order of drug administration was random. All drugs were applied extraluminally. Measurements were always taken 2 to 3 minutes after the drug was administered, when the response had stabilized. The vessels were washed three times with Krebs' buffer solution and allowed to equilibrate in a drug-free Krebs' buffer solution for 10 to 15 minutes between pharmacologic interventions.

Evaluation of Myogenic Contraction
After the vessels were equilibrated for at least 30 minutes at a pressure of 50 mm Hg, the active pressure-diameter relation was studied. Initially, the pressure was reduced to 10 mm Hg and the vessel was allowed to stabilize for 5 to 10 minutes. Then, the pressure was increased in increments of 10 mm Hg up to 100 mm Hg. At each pressure increment, changes in internal diameter were measured when the vessel response was stabilized (2 to 3 minutes). Upon completion of determination of the active pressure-diameter relation, the pressure was returned to 50 mm Hg. Papaverine (10^-4 mol/L) was then applied in the organ bath and the passive pressure-diameter relation was examined according to the above protocol.

Drugs
Isoproterenol, U46619, and sodium nitroprusside were obtained from Sigma Chemical (St. Louis, MO). Papaverine was obtained from Eli Lilly and Company (Indianapolis, IN). Forskolin and clonidine were obtained from RBI (Natick, MA). Forskolin was dissolved in dimethyl sulfoxide. Other drugs were dissolved in ultrapure distilled water. All solutions were prepared on the day of the study.

Data Analysis
The response of microvessels to each agent or pressure intervention was examined only once in each animal. The data were pooled from each dose-response in each experimental group and an average was calculated. The relaxation responses were expressed as percent relaxation of the U46619-induced precontraction of the vessel diameter. The diameter of each vessel in the myogenic experiments was normalized to the diameter at the pressure of 50 mm Hg in the presence of papaverine. Significance of the upward shift of the pressure-diameter relation was compared at the pressure range of 10 to 100 mm Hg. Differences were compared between groups as well as within the group before and after the application of papaverine. Significance of the differences in the shift in the pressure-diameter relation and of in vitro dose-responses of all experimental groups was determined by two-factor analysis of variance for repeated measures. Fisher's least significant difference multiple range test was used to make comparisons between groups when appropriate. Hemodynamic and blood flow data were analyzed with paired (two-tailed) Student's t test.

All values are expressed as mean ± standard error of the mean. A p value of less than 0.05 was considered to be significant.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Conclusion Acknowledgments References

 
Coronary Blood Flow and Myocardial Function
EARLY RESPONSE TO INTRACORONARY ISOPROTERENOL (DIRECT VASCULAR EFFECTS).
Baseline coronary blood flow in the left anterior descending artery, myocardial contractility, and in vivo responses to intracoronary isoproterenol administration are shown in Table 1. The early responses of CBF were recorded at approximately 20 seconds after initiation of drug administration, and heart rate, left ventricular fractional shortening, and dP/dt were unchanged. Thus, the early response is an indication of the direct vascular effect of the intracoronary infusion of isoproterenol. Before CPB and cardioplegic arrest, isoproterenol increased CBF by 94%. Five minutes after initiation of reperfusion in the Cryst CP-Rep group, while coronary blood flow was still elevated, isoproterenol increased CBF by 14% of the respective baseline level. In the Blood CP-Rep group, coronary blood flow increased by 37% of the respective baseline level 5 minutes after initiation of reperfusion. After 1 hour of postcardioplegia reperfusion, while coronary blood flow was also still elevated, blood flow responses to isoproterenol were similarly enhanced in the Cryst CP (76%) and Blood CP (67%) groups. Baseline CBF, left ventricular fractional shortening, and dP/dt were similar in pigs having undergone blood or crystalloid cardioplegia at the respective time points.

Vessel Characteristics
Coronary microvessels averaged 133 ± 7 µm, 130 ± 6 µm, 127 ± 6 µm, 128 ± 9 µm, and 133 ± 7 µm (internal lumen diameter) in the control, Cryst CP, Blood CP, Cryst CP-Rep, and Blood CP-Rep groups, respectively. Percent precontraction after application of U46619 was 36% ± 4%, 36% ± 5%, 35% ± 4%, 32% ± 6%, and 33% ± 3% in the control, Cryst CP, Blood CP, Cryst CP-Rep, and Blood CP-Rep groups, respectively. Mean concentrations of U46619 required to obtain these percent contractions were 10^-6.3 mol/L, 10^-6.3 mol/L, 10^-6.4 mol/L, 10^-6.1 mol/L, and 10^-6.1 mol/L in the control, Cryst CP, Blood CP, Cryst CP-Rep, and Blood CP-Rep groups, respectively.

In Vitro Vascular Studies
IN VITRO RESPONSE TO ß-ADRENERGIC AND ADENYLATE CYCLASE STIMULATION.
Isoproterenol induced a significant relaxation in control microvessels. The relaxation response to the ß-adrenoceptor agonist isoproterenol was reduced after either Cryst CP (p < 0.01 versus control) or Blood CP (p < 0.01 versus control), but to a greater extent after Cryst CP (p < 0.05 versus Blood CP). The response was significantly but not completely recovered after 1 hour of reperfusion in both groups (p < 0.01 Cryst CP-Rep or Blood CP-Rep versus control). In addition, the recovery in the Blood CP-Rep group was similar to that in the Cryst CP-Rep group (p = 0.82, Blood CP-Rep versus Cryst CP-Rep) (Fig 1).

View larger version (27K): [in this window] [in a new window]   Fig 1. . In vitro responses of coronary microvessels to the ß-adrenergic agonist isoproterenol. Vessels were harvested from control pigs (n = 6), pigs after 1 hour of crystalloid cardioplegia (Cryst CP) (n = 6) or blood cardioplegia (Blood CP) alone (n = 6), or after crystalloid (Cryst CP-Rep, n = 6) or blood cardioplegia (Blood CP-Rep, n = 6) and 1 hour of normothermic blood reperfusion. (p < 0.05 all groups versus control; p < 0.05 Blood CP versus Cryst CP; p = 0.84 Blood CP-Rep versus Cryst CP-Rep.)

View larger version (21K): [in this window] [in a new window]   Fig 2. . In vitro responses of coronary microvessels to the adenylate cyclase activator forskolin. Vessels were harvested from control pigs (n = 6), pigs after 1 hour of crystalloid cardioplegia (Cryst CP, n = 6) or blood cardioplegia (Blood CP) alone (n = 6), or pigs after crystalloid (Cryst CP-Rep, n = 6) or blood cardioplegia (Blood CP-Rep, n = 6) and 1 hour of normothermic blood reperfusion. (p < 0.01 Cryst CP versus control; p = 0.76 Blood CP versus control; p < 0.01 Cryst CP versus Blood CP; p < 0.05 Cryst CP-Rep versus Blood CP-Rep.)

₂-adrenoceptor-mediated and guanylate cyclase-mediated relaxation.

View larger version (23K): [in this window] [in a new window]   Fig 3. . In vitro responses of coronary microvessels to the endothelium-dependent 2-adrenergic agonist clonidine. Vessels were harvested from control pigs (n = 6), pigs after 1 hour of crystalloid cardioplegia (Cryst CP) (n = 6) or blood cardioplegia (Blood CP) alone (n = 6), or pigs after crystalloid (Cryst CP-Rep, n = 6) or blood cardioplegia (Blood CP-Rep, n = 6) and 1 hour of normothermic blood reperfusion. (p < 0.01 all groups versus control; p < 0.05 Cryst CP versus Blood CP; p < 0.05 Blood CP-Rep versus Cryst CP-Rep.)

View larger version (26K): [in this window] [in a new window]   Fig 4. . In vitro responses of coronary microvessels to the endothelium-independent vasodilator sodium nitroprusside. Vessels were harvested from control pigs (n = 6), pigs after 1 hour of crystalloid cardioplegia (Cryst CP) (n = 6) or blood cardioplegia (Blood CP) alone (n = 6), or pigs after crystalloid (Cryst CP-Rep, n = 6) or blood cardioplegia (Blood CP-Rep, n = 6) and 1 hour of normothermic blood reperfusion.

View larger version (22K): [in this window] [in a new window]   Fig 5. . Active pressure-diameter relations in porcine coronary arterioles from noninstrumented control animals (n = 6), pigs after 1 hour of crystalloid cardioplegia (Cryst CP) (n = 6) or blood cardioplegia (Blood CP) alone (n = 6), or after crystalloid (Cryst CP-Rep, n = 6) or blood cardioplegia (Blood CP-Rep, n = 6) and 1 hour of reperfusion. Vessel diameters were normalized to diameters at pressure of 50 mm Hg after application of papaverine. (p < 0.05 all groups versus control; p < 0.05 Blood CP versus Cryst CP.)

View larger version (20K): [in this window] [in a new window]   Fig 6. . Passive pressure-diameter relations in porcine coronary arterioles from noninstrumented control animals (n = 6), pigs after 1 hour of crystalloid cardioplegia (Cryst CP) (n = 6) or blood cardioplegia (Blood CP) alone (n = 6), or after crystalloid (Cryst CP-Rep, n = 6) or blood cardioplegia (Blood CP-Rep, n = 6) and 1 hour of reperfusion. Vessel diameters were normalized to diameters at pressure of 50 mm Hg after application of papaverine (Pap). (p < 0.05 all groups versus control.)

The decreased ß-adrenoceptor- and cyclic adenosine monophosphate-mediated responses during CPB and cardioplegic arrest have been demonstrated in cardiac myocytes and lymphocytes [15] and more recently in the coronary microcirculation [5]. The present study compares the effects of blood cardioplegia to crystalloid cardioplegia and the physiologic consequences (or lack) of the two strategies of myocardial protection. The greater reduction in relaxations to both isoproterenol and forskolin after crystalloid compared to blood cardioplegia suggests that a differential reduction in the activity of adenylate cyclase may be responsible for the altered profile of relaxation to a ß-adrenergic agonist observed in the present study. Adenylate cyclase activity may be affected by the oxidative stress of the myocardium during cardioplegia and oxygenated blood cardioplegia may better preserve the pH of myocardium during ischemia than crystalloid cardioplegia. However, increased oxidative stress actually increases rather than decreases the activity of adenylate cyclase [16]. Thus, it is unlikely that reduced oxidative stress is the cause of the improved activity of adenylate cyclase in the Blood CP group. Therefore, other characteristics or properties of blood or hemoglobin are likely involved in the preservation of ß-adrenergic responses in the coronary vessels.

Hemoglobin has pharmacologic properties that may alter vascular function and tone. It binds avidly to nitric oxide, which may impair endothelium-dependent relaxation and decrease the dilator effects of the basal release of nitric oxide from the endothelium. Nitric oxide in the presence of superoxide anion may produce the peroxynitrite radical, which can cause myocardial and vascular dysfunction during ischemia [17]. Nitric oxide bound to hemoglobin may not be available for this conversion to peroxynitrite during ischemia or cardioplegia. In addition, hemoglobin has also been found to interfere with sympathetic neural signal transduction [18]. Hemoglobin activates phospholipase C, which increases intracellular calcium concentration [19] by increasing intracellular levels of inositol-1,4,5-triphosphate and may cause the activation of protein kinase C. This may explain the increased myogenic contraction observed in vessels exposed to blood than those exposed to crystalloid cardioplegia. Finally, hemoglobin is a potent scavenger of oxygen-derived free radicals, which may damage cell membranes during cardioplegia or CPB [10].

Catecholamine-induced desensitization of ß-adrenoceptors is likely in part responsible for the reduced adrenergic signal transduction in the coronary circulation after CPB and cardioplegia. CPB and cardioplegia lead to increased circulating levels of catecholamines [2, 3]. Continuous stimulation of ß-adrenergic receptors causes phosphorylation by protein kinases including BARK (ß-adrenergic receptor kinase) and other less specific protein kinases. In turn, this causes the receptors to bind to arrestin, which translocates the receptor to the interior of the cell, in addition to inactivating the receptor [20]. Indeed, normothermic CPB is associated with reduced activity of ß-adrenergic receptors [21]. Extracorporeal circulation is also associated with increased capillary leakage and elevations of circulating levels of tumor necrosis factor and other inflammatory cytokines [22]. This may denature proteins and phospholipids in the cell membrane causing receptor dysfunction and impair signal transduction. In summary, the reason for the increased preservation of activity of ß-adrenergic receptors and adenylate cyclase after blood cardioplegia is not certain, probably mediated through several mechanisms, and cannot be discerned from the findings of the present study.

Activation of ₂-adrenoceptors on the endothelial cells has been demonstrated to relax vascular smooth muscle through the release of nitric oxide and the intracellular cyclic guanosine monophosphate pathway [5, 12]. The relaxation response to clonidine was significantly reduced by crystalloid cardioplegia and reperfusion, whereas the response after blood cardioplegia was better preserved. Relaxation to sodium nitroprusside was preserved, suggesting a selective injury to the endothelium. Previous studies have reported that cardioplegic arrest and reperfusion impair the coronary relaxations to bradykinin, serotonin, and other endothelium-dependent vasodilators [6–10], likely due in part to free radical scavenging properties of blood and hemoglobin. Thus, endothelial dysfunction is probably the mechanism underlying the reduced relaxation to ₂-adrenoceptor activation by clonidine.

Physiologic and Clinical Implications
It is important to note that although there was a clear difference in vascular preservation of in vitro response to ß-adrenergic, myogenic tone, and endothelium-dependent ₂-adrenergic stimulation in this study between blood and crystalloid cardioplegia, no difference was noted in the return of myocardial function, pressure development, or baseline CBF. Other investigators have demonstrated little benefit of blood cardioplegia in the nonischemic, noninjured heart in trials with patients [23, 24]. Thus, the clinical impact of preserved adrenergic and myogenic vasoregulation is uncertain. Furthermore, the advantage of improved preservation of ß-adrenergic-mediated responses after blood cardioplegia is likely of short duration. However, the altered myocardial perfusion and episodic coronary spasm observed in patients after cardiopulmonary bypass might be due in part to depression of ß-adrenoceptor- and cyclic adenosine monophosphate-mediated relaxations and to impaired endothelial function.

Myogenic contraction is involved in the autoregulation of coronary perfusion and blood flow to other organs. A decrease in myogenic contraction and intrinsic vascular tone of peripheral blood vessels may account for the low peripheral vascular resistance observed after CPB. Although at first thought it would seem that coronary arteriolar dilatation would have a positive effect on myocardial functional recovery, metabolic and autoregulatory function of the vascular system would be impaired, and blood flow over what would be required for metabolic needs may predispose to increased myocardial edema. Furthermore, mechanisms compensating for the reduced myogenic tone (eg, increased activity of phospholipase C) may lead to vascular contraction or spasm in the recovering vascular smooth muscle.

	Conclusion

Top Footnotes Abstract Introduction Material and Methods Results Conclusion Acknowledgments References

 
Hyperkalemic cardioplegia may interfere with normal ß-adrenergic signal transduction and myogenic contraction in the coronary circulation. It is unlikely that the addition of blood to the solution offers a clinically significant benefit over the use of crystalloid cardioplegia in limiting the impairment in these mechanisms of vascular smooth muscle regulation. However, a theoretical benefit may exist, as blood cardioplegia improves ₂-adrenoceptor-mediated relaxation and relaxations to other endothelium-dependent vasodilators. Whether improved endothelial preservation results in better long-term functional recovery of the heart and improved vascular health remains to be determined.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Conclusion Acknowledgments References

 
This study was supported by grant HL 46716 from the National Heart, Lung, and Blood Institute.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Conclusion Acknowledgments References

 
Address reprint requests to Dr Sellke, Division of Cardiothoracic Surgery, Beth Israel Hospital, Dana 905, 330 Brookline Ave, Boston, MA 02215.

	References

Top Footnotes Abstract Introduction Material and Methods Results Conclusion Acknowledgments References

 

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