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Cardiovascular Research 1997 36(2):223-235; doi:10.1016/S0008-6363(97)00176-4
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

β-Adrenergic signal transduction and contractility in the canine heart after cardiopulmonary bypass1

Jean-Yves Dupuisa,*, Kai Lic,2, Angelino Calderoned, Hugues Gosselinc, Xiao-Ping Yangd,3, Madhu B Anand-Srivastavad, Javier Teijeirab and Jean-Lucien Rouleauc

aDepartment of Anaesthesia, University of Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
bDepartment of Surgery, University of Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
cDepartment of Medicine, Montreal Heart Institute, 5000 Bélanger Est, Montréal, Québec, Canada, H1T 1C8
dDepartment of Physiology, University of Montreal, C.P. 6128, Montréal, Québec, Canada, M5B 1W8

* Corresponding author. Tel. (+1-613) 7614379; Fax (+1-613) 7614925; E-mail jdupuis@heartinst.on.ca

Received 11 December 1996; accepted 26 June 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Impaired β-adrenergic signal transduction has been proposed as a mechanism contributing to myocardial depression after cardiac surgery. This study determined the changes in the β-adrenergic system in a model of postoperative myocardial dysfunction induced by myocardial ischaemia and reperfusion under cardiopulmonary bypass (CPB). Those changes were then related to contractility and responsiveness to β-adrenergic stimulation. Methods: Four groups of dog hearts were studied: 7 hearts harvested immediately after anaesthesia induction (control group representing the preoperative cardiac condition); 6 hearts harvested after three hours of chest opening by sternotomy (open chest group serving as control for the effects of anaesthesia and surgery); 7 hearts harvested during CPB after 30 minutes of global ischaemia (ischaemia group); and 10 hearts from dogs submitted to one hour of CPB involving 30 minutes of global cardiac ischaemia, harvested 30 minutes after CPB (ischaemia–reperfusion group). Myocardial membranes were prepared to assess: (1) β-adrenergic receptor density using the radioligand [125I]iodocyanopindolol; (2) GTP-sensitive adenylate cyclase activity and its regulation by isoprenaline and forskolin; (3) G protein levels, using an immunoblotting technique. Ventricular trabeculae or papillary muscles served to assess contractility and responsiveness to isoprenaline. Results: The control and open chest groups had comparable β-adrenergic receptor density, adenylate cyclase activity and cardiac contractility. In the ischaemia group, the left ventricular membranes had a 55% decrease in receptor density as compared to the controls (P<0.005), similar GTP-sensitive adenylate cyclase activity and significantly lower adenylate cyclase responses to stimulation with isoprenaline and forskolin. In the ischaemia–reperfusion group, a 144% increase in the left ventricular receptor density was found as compared to the controls (P<0.005), with a 70% increase in GTP-sensitive adenylate cyclase activity (P<0.05), a similar adenylate cyclase response to isoprenaline and a 61% increase in response to forskolin (P<0.005). As compared to the controls, the ischaemia and ischaemia–reperfusion groups had comparable Gs{alpha} levels, but markedly decreased Gi{alpha}–2 and Gi{alpha}–3 levels. The baseline tension of the isolated muscles in the ischaemia and ischaemia–reperfusion groups was comparable, but was 61% and 47% lower than the controls, respectively (P<0.05). The maximal isoprenaline stimulated tension in the ischaemia and ischaemia–reperfusion groups was 66% and 36% lower than the controls, respectively (P<0.05 between all groups). Conclusions: The β-adrenergic system is severely depressed during global cardiac ischaemia under CPB, but recovers to supranormal values after CPB. However the increased cAMP generation by myocardial membranes after CPB is associated with decreased tension generation by corresponding cardiac muscles. Thus decreased contractility after CPB may be better explained by cellular alterations distal to cAMP generation rather than by changes in the β-adrenergic system.

KEYWORDS Beta-adrenergic receptor; Contractility; Cardiopulmonary bypass; Cyclic AMP; G proteins; Dog, anesthetized; Dog, ventricle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Myocardial depression frequently occurs after cardiac surgery involving cardiopulmonary bypass (CPB) [1]. In some cases the myocardial depression can be explained by an underlying preoperative ventricular dysfunction. However, in many cases it is thought to be the result of functional and biochemical alterations induced by procedure-related myocardial ischaemia–reperfusion injury [2, 3]. Desensitization of the β-adrenergic system during and after CPB has been suggested as one of the mechanisms contributing to myocardial depression after cardiac surgery [4, 5]. Evidence supporting this hypothesis has come from patients where drugs known to bypass the β-adrenergic signal transduction pathway (phosphodiesterase inhibitors) improved the cardiac performance after failure of response to maximal β-adrenergic receptor stimulation [6, 7]. More recently, uncoupling of the β-adrenoreceptors from the G-protein-adenylate cyclase complex was demonstrated during CPB in healthy canine hearts and in right atrial appendages from children with congenital heart diseases [4, 5]. In addition, mild downregulation of the β-adrenergic receptors was shown in the canine myocardium after separation from CPB [5]. These changes are attributed to the high levels of circulating catecholamines which have been consistently observed during and after CPB [4, 5]. However other factors related to cardiac surgery may also affect the β-adrenergic system. Reperfusion of the globally ischaemic heart following the release of the occlusive aortic cross-clamp may be associated with increased β-adrenergic receptor density and normal or increased adenylate cyclase activity, a combination which may result in greater β-agonist-induced production of cyclic adenosine monophosphate (cAMP) [8, 9]. The relative contribution of exposure to high levels of circulating catecholamines and ischaemia–reperfusion to changes in β-adrenergic signal transduction after CPB is unknown. Furthermore, the changes in the β-adrenergic system after CPB have not been correlated with cardiac function and their contribution to postoperative myocardial dysfunction remains to be determined.

This study was undertaken to better elucidate some of the pathophysiological mechanisms which may contribute to the development of myocardial depression after global ischaemia and reperfusion of the heart in the context of cardiac surgery with CPB. The study was designed with the assumption that global myocardial ischaemia and reperfusion during CPB are major contributing factors to postoperative myocardial depression [1]. Based on previous findings [4, 5], the main hypothesis was that a decrease in β-adrenergic signal transduction would occur after CPB and that it would contribute to myocardial depression and decreased contractile response to β-adrenergic stimulation after cardiac surgery. The objectives were to determine the changes in β-adrenergic signal transduction during and after cardiac surgery and relate these changes to cardiac contractility and responsiveness to β-adrenergic stimulation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 In vivo animal preparation and experimentation
The protocol was approved by the Institutional Animal Research Ethics Committee. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals". First, preliminary studies were conducted in 14 mongrel dogs in order to define a model which, after a period of global cardiac ischaemia without cardioprotection under CPB, would consistently demonstrate significant left ventricular depression 30 minutes after CPB, but would not require inotropic support for separation from CPB. The use of catecholamine inotropes, particularly the β-adrenergic agonists, even for a short duration was not desirable because these drugs may desensitize the β-adrenergic signal transduction pathway, an effect which could have confounded the results of the study [10]. Periods of global cardiac ischaemia of 30, 45 and 60 minutes followed by periods of reperfusion of similar duration during CPB were assessed. Thirty minutes of global cardiac ischaemia followed by a 25 minutes period of reperfusion during CPB reliably led to the desired model of postoperative left ventricular dysfunction. In accordance with the assumption that myocardial ischaemia and reperfusion are major contributors to postoperative myocardial depression [1], no cardioplegic solution was used in this model simulating the worst possible clinical scenario of poor intraoperative myocardial protection.

Thirty-five mongrel dogs weighing 29.1±1.1 kg were used in the experimental study. They were divided into four groups of which three were used to determine the time-course of the biochemical and functional changes in the myocardium throughout the perioperative period of cardiac surgery with CPB and one served as control for the effects of the in vivo experimentation without CPB. In the first group, twelve normal dogs had their heart harvested through a left thoracotomy immediately after induction of anaesthesia. Left thoracotomy is the most rapid approach to harvest the canine heart in our laboratory with the lowest risk of procedure related myocardial damages. Therefore those hearts were the best estimate of the preoperative myocardial condition and served as true controls for in vitro biochemical and mechanical studies (control group). Six dogs formed a second group which served as controls for the effects of anaesthesia, chest opening and surgical time on the myocardium: their chest was opened through a sternotomy and left open after instrumentation for three hours before harvesting the heart (open chest group). The third group counted seven dogs which served to assess the intraoperative cardiac condition during peak myocardial depression: they had a sternotomy and were submitted to 30 minutes of global myocardial ischaemia during CPB, with no reperfusion before harvesting the heart (ischaemia group). Finally, ten other dogs were used to determine the cardiac condition in the early postoperative period: they were submitted to one hour of CPB which involved 5 minutes of total body cooling followed by 30 minutes of global myocardial ischaemia induced by cross-clamping the aorta and 25 minutes of myocardial reperfusion; the heart of these dogs were harvested 30 minutes after separation from CPB (ischaemia–reperfusion group).

All dogs were anaesthetized with intravenous pentobarbital (5 mg/kg), fentanyl (50 µg/kg), diazepam (0.2 mg/kg) and pancuronium (0.15 mg/kg). Mechanical ventilation was established through an endotracheal tube using 100% oxygen with a minute volume which provided arterial pH of 7.30 to 7.45 and an arterial PCO2 of 30 to 45 mmHg. Anaesthesia was maintained with intermittent boluses of fentanyl (10 µg/kg) and diazepam (0.2 mg/kg) given every 30 to 40 minutes or more often if necessary in order to keep the mean arterial pressure between 70 and 80 mmHg. This high-dose opioid anaesthesia protocol was selected because of its similarity with techniques used in cardiac surgical patients, its minimal effect on myocardial function and its efficacy in blunting the haemodynamic and metabolic responses to noxious stimuli [11, 12]. However like any other anaesthesia technique, high-dose fentanyl anaesthesia does not prevent increases in plasma catecholamines associated with the use of CPB [12].

Following induction of anaesthesia, the dogs in the open chest, ischaemia and ischaemia–reperfusion groups were monitored with an electrocardiogram (lead II). Insertion of an arterial line through the right carotid artery was performed. A pulmonary artery catheter allowing measurement of cardiac output (CO) by thermodilution (American Edwards Laboratory, model 9520 A, Irvine, CA) was inserted via the right internal jugular vein. The carotid and pulmonary artery catheters were attached to a pressure transducer (Cobe, model 042-982-100, Lakewood, CO) and a pressure processor (Gould, model 13-4615-52, Cleveland, OH) for monitoring of systemic and pulmonary artery pressures. The chest was opened through a sternotomy and an adjustable constricting ring was placed around the descending aorta. A pericardial cradle was formed. An intraventricular catheter tip manometer (MPC 500; Millar Instrument Inc., Houston, TX) was inserted in the left ventricle via the apex and connected to a pressure processor and differentiator (Gould, model 13-4615-71, Cleveland, OH) for continuous measurement of the ventricular pressure and its first derivative (dP/dt).

Once the animal preparation was complete (approximately one hour after sternotomy), the heart rate (HR) was maintained between 120 and 130 beats/minute using right atrial pacing. Left ventricular end-diastolic pressure (LVEDP) was increased and maintained at 10 mmHg by volume loading with 0.9% NaCl solution and the mean arterial (MAP) was increased and maintained around 140 mmHg by constriction of the descending aortic ring. After a period of stabilization of approximately 5 minutes with these preload and afterload conditions, and atrial pacing, myocardial function was assessed by measuring three variables: (1) CO which was the average of three measurements with less than a 10% difference; (2) left ventricular stroke work (LVSW) as calculated with the following formula: LVSW=(MAP–LVEDP)x(CO/HR)x0.0136; (3) positive and negative left ventricular dP/dt. The aortic ring was released and the atrial pacemaker turned off during the intervals between the assessments of cardiac function.

The in vivo evaluation was repeated every 30 minutes for 3 hours in the open chest group. The hearts were then harvested for in vitro biochemical and mechanical studies. In the ischaemia and ischaemia–reperfusion groups, following the first evaluation of myocardial function, intravenous heparin 400 units/kg was given. An arterial cannula was inserted in the left femoral artery and connected to the CPB circuit. A right atrial cannula was inserted and connected to the venous reservoir of the CPB which was primed with 1.5 l of 0.9% NaCl solution. A bubble oxygenator and a roller pump providing nonpulsatile blood flow were used for CPB. The flow was started at 100 ml/kg and subsequently readjusted in order to maintain normal acid–base balance and mean arterial pressure between 60 and 80 mmHg. Mechanical ventilation was discontinued during CPB. Moderate hypothermia (rectal temperature of 30 to 32°C) was induced within five minutes of CPB initiation and maintained until the end of the period of global cardiac ischaemia. A vent was inserted through the left ventricular apex. Global myocardial ischaemia was induced by cross-clamping the aorta while decompressing the left ventricle through the vent. The hearts were totally arrested within 4 to 7 minutes. After 30 minutes of ischaemia, the hearts in the ischaemia group were harvested without reassessing their in vivo function as they were asystolic. In the ischaemia–reperfusion group, the aortic cross-clamp was released and active rewarming was started to reach a rectal temperature of 37°C before separation from CPB. At the return of electrical activity, the hearts were internally defibrillated with 10 to 20 J. Haematocrit was kept between 0.21 and 0.24 during CPB and increased to at least 0.28 at the end of CPB, using blood transfusions when necessary. After 25 minutes of reperfusion the dogs were weaned from CPB and stabilized with volume at a LVEDP of 10 mmHg. No inotropic agent was given. The atrial cannula was removed and the in vivo evaluation of the cardiac function was repeated 30 minutes following separation from CPB. The hearts were then harvested for in vitro studies.

Arterial blood gases were measured routinely throughout the experiments to assure normal acid–base balance. Venous blood samples (5 ml) were collected from the proximal port of the pulmonary artery catheter and mixed with 100 µl of 0.25 mol/l EGTA and 0.2 mol/l glutathione (pH 7) for measurements of plasma catecholamine levels. Measurements of catecholamines were done in the ischaemia and ischaemia–reperfusion groups at the following time points: A, before skin incision; B, after sternotomy; C, after completion of the animal preparation; D, after 5 minutes of CPB, immediately before cross-clamping the aorta; E, at the end of the myocardial ischaemic period. There were two additional time points for measurements of catecholamines in the ischaemia–reperfusion group: F, at the end of CPB; G, 30 minutes after separation from CPB. In the open chest group, the samples were collected at time points A, B, C and at time intervals corresponding with the time points D, E, F and G in the ischaemia–reperfusion group. The blood samples were kept at 4°C until the end of the experiment. They were then centrifuged at the same temperature and one-ml plasma samples were mixed with 20 µl of 0.25 mol/l EGTA and 0.2 mol/l glutathione (pH 7) and stored at –80°C for later analysis. Plasma catecholamine concentrations were determined by the radioenzymatic assay of Peuler and Johnson [13], with minor modifications, including addition of 2 N perchloric acid (1:9) to deproteinize the plasma and a doubling of the buffer capacity of the reactive mixture.

After removal from the chest, all hearts were immediately placed in oxygenated Krebs–Henseleit solution at 4°C. While immersed in this solution the left and right ventricles were opened. Within 10 minutes, one or more papillary muscles and/or trabeculae were removed for the mechanical studies; the remaining tissue was used for preparation of myocardial membranes necessary for biochemical analysis.

2.2 Preparation of myocardial membranes
Two gram tissue samples isolated from the left and right ventricular free walls were minced in 30 ml of buffer A, which contained 0.25 mol/l sucrose, 5 mmol/l Tris-HCl, 1 mmol/l MgCl2, 1 mmol/l EDTA, and 10 µmol/l phenylmethyl-sulfonyl fluoride (PMSF, pH 7.4) at 4°C. The samples were homogenized with three bursts of 5 to 7 seconds in a polytron (Brinkmann T, Brinkmann Instruments, Inc., Westbury, N.Y.) at maximum speed. The homogenate was filtered through three layers of cheesecloth and centrifuged at 1000 g for 10 minutes at 4°C. The supernatant was removed and centrifuged at 45 500 g at 4°C for 25 minutes. The supernatant was discarded, and the pellet was resuspended in 30 ml buffer B, which contained 50 mmol/l Tris-HCl, 10 mmol/l MgCl2, 1 mmol/l EDTA, and 10 µmol/l PMSF, with five strokes in a 40-ml dounce type putter and recentrifuged at 45 500 g at 4°C for 25 minutes. This step was repeated twice, and the pellet was resuspended in an appropriate volume of buffer B to obtain a protein content of 1 mg/ml. Protein content was determined by the method of Lowry et al. [14]. The membrane preparations were kept at –80°C for later analysis of radioligand binding, measurement of adenylate cyclase activity and quantitative immunoblotting of the guanine nucleotide binding proteins (G proteins).

2.3 β-adrenergic receptor radioligand binding studies
Before the assay, the membrane preparations were thawed and homogenized. The β-adrenergic receptor studies were done with the radioligand [125I]iodocyanopindolol ([125I]CYP). Binding of β-adrenergic receptors with [125I]CYP was measured by incubating 10 µl membrane preparation (1 mg/ml) with eight concentrations of radioligand (10 to 300 pmol) at 25°C for 2 hours in a total volume of 250 µl. Nonspecific binding was determined in the presence of the β-antagonist alprenolol (20 µmol). The incubations were terminated by a rapid dilution of the assay mixture with 1 ml cold buffer B, followed by a rapid vacuum filtration through Whatman GF/C glass fiber filters (Whatman Inc., Clifton, NJ). The filters were rapidly washed four times using 3-ml aliquots of cold buffer B. After the filters were dried, the radioactivity was counted in 5 ml scintillation fluid (liquofluor PRO-POPOP toluene concentrate, New England Nuclear). The total β-adrenergic receptor population was defined as the difference between the total amount of radioactivity bound in the presence of [125I]CYP alone and the nonspecific binding in the presence of [125I]CYP and 20 µmol unlabeled (–)-alprenolol. The specific binding routinely obtained was 80%. Receptor density and the equilibrium dissociation constant Kd for [125I]CYP were determined by non-linear regression analysis (All Fit).

2.4 Measurement of adenylate cyclase activity
After thawing and homogenization of the membrane preparations, cAMP formation was assessed by measuring the conversion of 32P-ATP (New England Nuclear) to 32P-cAMP, as previously described [15]. Briefly, 30–40 µg of protein was added to a total volume of 50 µl containing 5 mmol/l Tris-HCl, 1 mmol/l MgCl2, 0.1 mmol/l EDTA, 1 µmol/l PMSF, 0.12 mmol/l ATP, 1 mmol/l cAMP, 0.1 mmol/l GTP, 1 mmol/l isobutylmethylxanthine (IBMX), 2.7 mmol/l creatine phosphate, 0.1 mg/ml creatine kinase, and 1–2x106 cpm/assay of Formula . The reaction proceeded for 15 minutes at 37°C in the absence or presence of agonist. The Formula formed was isolated as previously described [16]. Adenylate cyclase activity was assessed in the presence of the direct activator forskolin at concentrations of 10–9 to 10–4 mol/l. Stimulation of the adenylate cyclase with isoprenaline at concentrations of 10–9 to 10–3 mol/l was performed in the presence of the antioxidant ascorbic acid (0.1 mmol/l).

2.5 Quantitative immunoblotting of the stimulatory guanine nucleotide protein (Gs) and of the inhibitory guanine nucleotide binding protein (Gi)
After sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the separated proteins were electrophoretically transferred to nitrocellulose paper (Schleicher and Schuell) as previously described [17], using a minitransfer apparatus (Bio-Rad) at 100 V for 1 hour or a semidry transblot apparatus (Bio-Rad) at 15 V for 45 minutes. After transfer, the membranes were washed twice in phosphate-buffered saline (PBS) and were incubated in PBS containing 3% BSA at room temperature for 2 hours. The blots were then incubated with antisera against G proteins (RM/1 against Gs{alpha}, AS/7 against Gi{alpha}–2 and EC/2 against Gi{alpha}–3) and in PBS containing 1% BSA and 0.1% Tween-20 at room temperature for 2 hours. The antigen–antibody complexes were detected by incubating the blots with goat anti-rabbit IgG (Bio-Rad) conjugated with horseradish peroxidase for 2 hours at room temperature. The blots were washed three times with PBS before reaction with enhanced chemiluminescence Western-blotting detection reagents from Amersham as previously described [18]. The autoradiograms and immunoblots were quantified by densitometric scanning using an enhanced laser densitometer (LKB Ultroscan XL, Pharmacia, Quebec, Canada) and gel scan XL evolution software (version 2.1, Pharmacia). The scanning was one dimensional and scanned the entire area of protein band in autoradiograms and immunoblots.

2.6 In vitro mechanical studies
Either left or right ventricular trabeculae or papillary muscles located in the basal portion of the free wall were dissected and mounted in an isolated bath of Krebs–Henseleit solution containing (mmol/l) NaCl 118, KCl 3.5, MgSO4 2.43, CaCl2 1.25, KH2PO4 1.2, NaHCO3 24.9 and dextrose 5% bubbled with 95% O2–5% CO2, at a pH of 7.4 and a temperature of 29°C. Only muscles free from the ventricular wall for at least 4 mm and with a cross-sectional area of <1.2 mm2 were used for the study. It has been shown in a previous study that there is no difference in total tension (T) or dT/dt between dog left and right ventricular muscles and that muscles from both ventricles respond similarly to β-adrenergic stimulation [19]. Seven muscles (2 from the left and 5 from the right ventricle) were obtained from the control dogs, 6 muscles from the open chest group (2 from the left and 4 from the right ventricle), 6 muscles (2 from the left and 4 from the right ventricle) from the ischaemia group, and 10 muscles (3 from the left and 7 from the right ventricle) were obtained from the ischaemia–reperfusion group. The base of the muscle was held by a stainless-steel clamp, and the other end was tied to a prototype lever with an electromagnetic feedback system identical to that described by Brutsaert et al. [20]. The instruments and protocol for muscle stimulation were previously described in detail [21]. The maximal intrinsic contractility of the muscles during isometric, isotonic and unloaded contractions was assessed by adding CaCl2 from a stock solution to the bath to achieve a 15 mmol/l concentration before and after stimulation with isoprenaline. The β-adrenergic responsiveness of the muscles was tested by building an isoprenaline concentration–response curve with isometric, isotonic and unloaded contractions measured at different concentrations (10–9 to 3x10–5 mol/l) in a bath containing 1.25 mmol/l CaCl2.

2.7 Data analysis and statistics
Tension generation by isolated myocardial muscles was used as the main marker of myocardial depression. Preliminary studies indicated that with a minimum of six isolated muscles in each group, a 30% reduction (standard deviation of 15%) in maximal tension generation could be expected with an alpha error of 0.05 and a power of 0.80. Thus a minimum of six sets of biochemical and mechanical studies were required in each group to complete this research. In three of the groups, more than six animals were studied either because of the absence of adequate trabeculae or papillary muscles in some hearts or because of accidental thawing of the myocardial membranes during transportation from one laboratory to the other. Values are expressed as mean±standard error of the mean (SEM). Quantitative data were compared between the four groups by one-way analysis of variance (ANOVA). This was followed by multiple unpaired Student's t-tests with Bonferroni correction when necessary. The effects of forskolin and isoprenaline on the cAMP production by the membrane preparations, and the effect of isoprenaline on the isometric, isotonic and unloaded contractions of the papillary muscles were compared by ANOVA for repeated measures. A Dunnett's t-test was used for analysis of repeated measures within a group. Statistical significance was determined by a P value <0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 In vivo experimentation
In the control group, the dogs were not submitted to any in vivo tests and were sacrificed immediately after induction of anaesthesia. In the other three groups (open chest, ischaemia and ischaemia–reperfusion), the controlled variables were successfully maintained near the targets of 120–130/min for HR, 140 mmHg for MAP and 10 mmHg for LVEDP during the in vivo evaluations of cardiac function. The three groups had comparable baseline left ventricular performance as determined by CO, LVSW, positive and negative left ventricular dP/dt (Table 1). All dogs in the ischaemia–reperfusion group were successfully weaned from CPB without the use of catecholamine inotrope. There was no change of left ventricular performance over time in the open chest group as determined by the in vivo indices of myocardial function used in this study. However, as desired for this experimental protocol all the hearts in the ischaemia–reperfusion group had significant myocardial depression after CPB as demonstrated by the values of CO, LVSW, positive and negative left ventricular dP/dt which were 38%, 45%, 20% and 27% lower than before CPB, respectively (P<0.05 for all measured variables).


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  		Table 1 Haemodynamics and left ventricular performance at baseline and at the end of the in vivo experiment

 
Immediately after induction of anaesthesia the plasma levels of noradrenaline and adrenaline in the open chest, ischaemia and ischaemia–reperfusion groups were comparable. These levels did not change significantly throughout the experiment in the open chest group (Fig. 1). In the ischaemia and ischaemia–reperfusion groups, the levels of noradrenaline and adrenaline remained comparable until the end of the cardiac ischaemic period (time point E), with no significant changes over time in either group up to that time point (Fig. 1). However, significant increases in the levels of noradrenaline and adrenaline were observed in the ischaemia–reperfusion group at the end of CPB (time point F) and 30 minutes after CPB (time point G).


Figure 1
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  		Fig. 1 Plasma catecholamine levels over time in 6 dogs exposed to cardiac exposure by sternotomy for three hours (open chest), 7 dogs exposed to global cardiac ischaemia under cardiopulmonary bypass (ischaemia) and 10 dogs exposed to global myocardial ischaemia and reperfusion (ischaemia–reperfusion) under cardiopulmonary bypass (CPB). Blood samples were collected at: A, before skin incision; B, after sternotomy; C, after completion of the animal preparation; D, after 5 minutes of CPB; E, after 30 minutes of CPB; F, at the end of CPB; G, 30 minutes after separation from CPB. In the ischaemia group, the hearts were harvested immediately after the time point E. In the open chest group, time intervals corresponding with the time points D, E, F and G in the ischaemia–reperfusion group were used. *P<0.05, as compared to value at time point A, within the ischaemia–reperfusion group; {dagger}P<0.05, as compared to open chest group at the corresponding time points.

 
3.2 β-Adrenergic receptor radioligand binding studies
Fig. 2 represents a plot of [125I]CYP binding to cardiac membranes from the left ventricle of dogs in the control and ischaemia–reperfusion groups. Total β-adrenergic receptor density in left ventricular membrane preparations, as measured by [125I]CYP binding in fmol/mg of protein, was 534±53 in the control group, 496±50 in the open chest group, 240±54 in the ischaemia group (P<0.005 versus control and open chest groups) and 1304±156 in the ischaemia–reperfusion group (P<0.005 versus all other groups). Similar results were found in the right ventricular membrane preparations where the total β-adrenergic receptor density in fmol/mg was 412±59 in the control group, 427±91 in the open chest group, 229±50 in the ischaemia group (P<0.05 versus control and open chest groups) and 737±54 in the ischaemia–reperfusion group (P<0.005 versus all other groups). No significant differences in β-adrenergic receptor density were found between the left and right ventricles within each group. No significant differences in Kd for [125I]CYP binding were found between the three groups in both ventricles. The Kd for [125I]CYP binding was also comparable between the left and right ventricles within each group.


Figure 2
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  		Fig. 2 A representative plot of [125I]iodocyanopindolol binding to cardiac membranes from the left ventricle of dogs in the control and ischaemia–reperfusion groups. Total β-adrenergic receptor density in this case was 109% higher in the ischaemia–reperfusion membranes obtained 30 minutes after cardiopulmonary bypass, as compared with the control membranes obtained immediately after anaesthesia induction.

 
3.3 Adenylate cyclase activity
Table 2 presents the adenylate cyclase activity in presence of the phosphodiesterase inhibitor IBMX (1 mmol/l) and GTP (0.1 mmol/l), as measured by the generation of cAMP in the membranes from the left and right ventricles before and after maximum stimulation with forskolin (0.1 mmol/l) and isoprenaline (1 mmol/l). The left ventricular membranes from the control, open chest and ischaemia groups had comparable adenylate cyclase activity in presence of GTP. The adenylate cyclase activity in the open chest group was slightly higher than in the control and the ischaemia groups, but the differences did not reach statistical significance (P = 0.19). In the ischaemia–reperfusion group, the differences were significant with an adenylate cyclase activity 70% and 75% higher than in the control and ischaemia groups, respectively (P = 0.025). After maximal stimulation of the left ventricular membranes with forskolin and isoprenaline, the cAMP levels in the control and open chest groups were comparable and markedly higher than in the ischaemia group. The cAMP levels after stimulation with those two agents in the ischaemia–reperfusion group were significantly higher than in all other groups. Similar quantitative and qualitative patterns of adenylate cyclase reactivity were observed in the right ventricular membranes from the four groups. Fig. 3 shows the adenylate cyclase activity in the left ventricular membrane preparations in response to stimulation with various concentrations of forskolin and isoprenaline. The concentration–response curves of adenylate cyclase activity with forskolin and isoprenaline were not significantly different between the control, open chest and ischaemia groups. In the ischaemia–reperfusion group, the concentration–response curves after stimulation with forskolin and isoprenaline were significantly higher than in the other three groups (P<0.005 versus each group).


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  		Table 2 Generation of cAMP in canine membrane preparations from the left and right ventricles in the presence of GTP before and after maximum stimulation with forskolin and isoprenaline

 

Figure 3
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  		Fig. 3 Adenylate cyclase response to various concentrations of forskolin and isoprenaline in membrane preparations from the left ventricles in the control (n = 7), open chest (n = 6), ischaemia (n = 6) and ischaemia–reperfusion (n = 8) groups. The control, ischaemia and ischaemia–reperfusion groups are representative of the preoperative, intraoperative and postoperative heart conditions, respectively. The open chest group served as control for anaesthesia and surgery time. The concentration–response curves for forskolin and isoprenaline were significantly higher in the ischaemia–reperfusion group than in the other three groups. ANOVA for repeated measures: *P<0.05.

 
The responses of the left and right ventricular membranes after stimulation with forskolin and isoprenaline were also analyzed in terms of maximum absolute increases of cAMP generation from values obtained in presence of GTP alone. After maximal stimulation of the left ventricular membranes with forskolin, the absolute increases of adenylate cyclase activity in pmol cAMP/mg of protein/min were 718±45 in the controls, 669±136 in the open chest group, 448±69 in the ischaemia group (P<0.01 versus control and open chest group) and 1159±85 in the ischaemia–reperfusion group (P<0.001 versus all other groups). After isoprenaline stimulation, the increases in pmol cAMP/mg of protein/min were 79±11 in the controls, 53±8 in the open chest group, 40±17 in the ischaemia group (p = 0.003 versus control) and 95±10 in the ischaemia–reperfusion group (P = 0.002 versus ischaemia only). Similar results were observed in the right ventricular membranes of each group.

3.4 Quantitative immunoblotting of Gs{alpha}, Gi{alpha}–2 and Gi{alpha}–3
In an attempt to explain the significant changes in adenylate cyclase activity found in the ischaemia and ischaemia–reperfusion groups, the left ventricular membrane preparations from 5 dogs in the control group, 5 dogs in the ischaemia group and 7 dogs in the ischaemia–reperfusion group were used to determine the G protein contents by immunoblotting technique (Fig. 4). The relative amount of immunodetectable Gs{alpha} as compared to the control group was 122±26% in the ischaemia group and 107±12% in the ischaemia–reperfusion group (P = N.S. between all groups). The levels of Gi{alpha}–2 in the ischaemia and ischaemia–reperfusion groups were 46±14% and 31±9% of the controls, respectively (p = 0.0001). The Gi{alpha}–3 levels were 68±14% of the controls in the ischaemia group (P = 0.04) and 78±14% of the controls in the ischaemia–reperfusion group (P = 0.07).


Figure 4
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  		Fig. 4 Autoradiograms of immunoblots representing the expression of stimulatory (Gs{alpha}) and inhibitory (Gi{alpha}–2 and G i{alpha}–3) G protein {alpha}-subunits in myocardial membranes from the control, ischaemia (ISCH) and ischaemia–reperfusion (ISCH Rep) groups. The membrane proteins (40 µg) from each group were resolved by SDS-PAGE, transferred to nitrocellulose, then immunoblotted using RM/1 antibody against Gs{alpha}, AS/7 against Gi{alpha}–2 and EC/2 against Gi{alpha}–3. The autoradiograms shown are representative of three or four separate experiments. The ischaemia (intraoperative) and ischaemia–reperfusion (postoperative) groups had lower levels of Gi{alpha}–2 and Gi{alpha}–3 than the control (preoperative) group.

 
3.5 Mechanical characteristics of the isolated trabeculae and papillary muscles
There was no significant difference in resting muscle length between the groups (control, 5.8±0.7 mm; open chest, 4.7±0.5 mm; ischaemia, 4.3±0.3 mm; ischaemia–reperfusion, 4.9±0.3 mm). The mean cross-sectional area was 0.8±0.2 mm2 in the open chest group and 0.7±0.1 mm2 in the other groups. At baseline, no significant differences in mechanical characteristics were found between the control and open chest groups. However muscle tension, positive and negative dT/dt, and time to peak tension were significantly lower in the ischaemia and ischaemia–reperfusion groups than in the control and open chest groups (Table 3). The baseline Vmax was lower in the ischaemia and ischaemia–reperfusion groups than in the control and open chest groups, but a significant difference was found only in the comparisons with the control group. At baseline, the muscles from the ischaemia and ischaemia–reperfusion groups had numerous comparable baseline characteristics including tension, positive dT/dt, negative dT/dt and Vmax. The only difference between these two groups was the greater time to peak tension in the ischaemia–reperfusion muscles.


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  		Table 3 Mechanical characteristics of canine papillary muscles and trabeculae at baseline and after stimulation with isoprenaline 30 µmol/l and calcium 15 mmol/l

 
After stimulation with isoprenaline, all mechanical characteristics in the ischaemia and ischaemia–reperfusion groups remained smaller than in the control and open chest groups (Table 3), but all variables were larger in the ischaemia–reperfusion group than the ischaemia group. Similar results were obtained when the effects of isoprenaline were presented as concentration–tension curves (Fig. 5). The control, open chest and ischaemia–reperfusion groups had comparable absolute changes in tension. These were significantly greater than in the ischaemia group.


Figure 5
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  		Fig. 5 Tension generation response of trabeculae and papillary muscles to isoprenaline expressed in absolute values and absolute increases of tension from baseline. Values are mean±SEM. Significant differences in the concentration–response curves were found between the groups. ANOVA for repeated measures: *P<0.05, ischaemia–reperfusion versus the other three groups; {dagger}P<0.05, ischaemia group versus the other three groups.

 
Stimulation with 15 mmol/l calcium (Table 3) increased tension generation by nearly 100% in all the groups. After calcium stimulation, all mechanical characteristics remained significantly higher in the control and open chest groups as compared to the other two groups, except for Vmax which became comparable between the control, the open chest and the ischaemia–reperfusion groups. All mechanical similarities observed at baseline between the muscles from the ischaemia and ischaemia–reperfusion groups were eliminated by calcium stimulation: tension, positive dT/dt, negative dT/dt and Vmax in the ischaemia–reperfusion group became significantly higher than in the ischaemia group.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
This study assesses myocardial contractility and the integrity of the β-adrenergic signal transduction pathway in a model of myocardial depression induced by global cardiac ischaemia under CPB, without cardioprotection. The assessment was performed 30 minutes following CPB. This time point represents the end of cardiac surgery, a period during which severe depression of left ventricular function is often observed [1]. It is also a time when hearts are commonly dependent on pharmacological inotropic support, thus increasing the interest of defining the characteristics of the β-adrenergic system at that time. The results of the study do not support the original hypothesis that a decrease in β-adrenergic signal transduction would contribute to myocardial depression and decreased contractile response to β-adrenergic stimulation after CPB with global myocardial ischaemia followed by reperfusion. Following CPB, decreased contractility in isolated cardiac muscles is associated with increased β-adrenergic receptor density and adenylate cyclase activity in corresponding membrane preparations. This major finding suggests that myocardial depression after CPB may be the result of abnormalities beyond the β-adrenergic signal transduction pathway. This contrasts with the findings in hearts exposed only to global ischaemia under CPB, where abnormalities in and beyond the β-adrenergic signal transduction are observed in association with severely depressed contractility of cardiac muscles. No changes in myocardial contractility and c-AMP generation are observed after a few hours of anaesthesia and heart exposure by sternotomy. Therefore CPB and its procedure-related myocardial ischemia–reperfusion injury are the major determinants of myocardial depression and changes in β-adrenergic signal transduction during and after CPB in this study.

In a previous evaluation of β-adrenergic signal transduction in canine hearts, Schwinn et al. also observed an absence of changes in myocardial β-adrenergic receptor density and adenylate cyclase activity after a few hours of cardiac exposure by sternotomy without CPB [5]. However in contrast with the present study, they found downregulation of the β-adrenergic receptors 30 minutes following CPB with global cardiac ischaemia and reperfusion. They attributed this result to cardiac exposure to increased levels of plasma catecholamines during and after CPB. The difference between their results and those in the present study may be explained by differences in experimental protocol. First, the duration of CPB in the study by Schwinn et al. was almost three times as long (155 minutes) as in this study, allowing a longer exposure to increased levels of plasma catecholamines. Second, in their study, ischaemic cardiac arrest was induced and maintained by cold potassium cardioplegia. In the present study, the use of cardioplegic solution was avoided to simulate a heart failure model resulting from poor intraoperative myocardial protection and reperfusion injury. This difference may be very important as increased β-adrenergic receptor densities and adenylate cyclase activity have been demonstrated during and after myocardial ischaemia in other experimental models [8, 9]. A third difference is that Schwinn et al. used transmyocardial biopsies with a Tru-cut biopsy needle. This limited the biochemical analyses to a small apical area which may not represent the state of the membranes in other parts of the heart. In addition to downregulation of the β-adrenergic receptors after CPB, Schwinn et al. found increased levels of cAMP after membrane stimulation with a β-adrenergic agonist. The difference with control levels of cAMP did not reach statistical significance as it did in the present study. However in presence of decreased receptor density, the results of Schwinn et al. can only be explained by an increase of adenylate cyclase activity following CPB, as found in the present study.

The exact mechanism by which cAMP generation is increased after CPB remains uncertain. This may be due to an intrinsic modification (e.g. phosphorylation) of the adenylate cyclase, or to altered interactions with the G proteins, or to a combination of both phenomena. In this study, maximal stimulation of the membranes with forskolin, a direct activator of adenylate cyclase, led to much greater increases of cAMP in the ischaemia–reperfusion group as compared to the other three groups. This suggests a greater efficacy of adenylate cyclase in the hearts from the ischaemia–reperfusion group and is consistent with the hypothesis of an intrinsic modification of adenylate cyclase after CPB. Adenylate cyclase activity may also be influenced by changes in the levels of Gs{alpha} or Gi{alpha}–2 and Gi{alpha}–3. It has previously been shown that the decreased expression of Gi{alpha}–2 is associated with a decreased inhibition of adenylate cyclase and a greater cAMP generation after stimulation of adenylate cyclase with agents like GTP and forskolin [17]. Since the expression of Gs{alpha} was comparable between the control and ischaemia–reperfusion groups, the decreased expression of Gi{alpha}–2 and Gi{alpha}–3 in the ischaemia–reperfusion group may also have contributed to the increased adenylate cyclase activity in this group.

As compared to the ventricular membranes in the control group, the left and right ventricles from the ischaemia–reperfusion group had a 144% and 79% increase in β-adrenergic receptor density, respectively. However, after maximal β-adrenergic stimulation the absolute increases of cAMP production by the left and right ventricular membranes in the ischaemia–reperfusion and control groups were comparable. The reason why the increased receptor density was not associated with a proportional absolute response to isoprenaline in the ischaemia–reperfusion group may only be explained by an uncoupling of the myocardial receptors from the Gs{alpha} protein. This early desensitizing phenomenon occurs within minutes of β-adrenergic stimulation [22]. Exposure to high levels of circulating catecholamines during and after CPB may have initiated this event. Downregulation of the receptor population was not observed in the ischaemia–reperfusion group as previously described [5]. This is probably because it is a late desensitizing process which requires 3 to 6 hours of exposure to catecholamines [22]. In this study, exposure to increased circulating catecholamines was less than one hour.

The GTP-sensitive adenylate cyclase activity was similar in the ischaemia and control groups despite significantly lower levels of Gi{alpha}–2 and Gi{alpha}–3 in the ischaemia group. In addition, stimulation of the cardiac membranes with forskolin in the ischaemia group led to much lower increases of cAMP production as compared to the control membranes. Those findings suggest a decreased efficacy of the adenylate cyclase in the ischaemia group. Furthermore a 55% and 44% decrease of β-adrenergic receptor density was found in the left and right ventricles of the ischaemia group, respectively. This decreased receptor density was accompanied by a proportional decrease in the production of cAMP after isoprenaline stimulation. These results demonstrate severe depression of the β-adrenergic signal transduction during global cardiac ischaemia under moderately hypothermic cardiopulmonary bypass. These findings contrast with those from models of regional myocardial ischaemia [9, 23]or unperfused isolated hearts [24]where increased receptor density and adenylate cyclase activity are commonly observed within 15 minutes of ischaemia. Whether the refined membrane preparations used in the present study caused a receptor loss by discarding the low speed supernatant is possible [25]. However the observed receptor densities were high in all groups and comparable to those found by other investigators using a similar membrane preparation and the same radioligand [26]. In addition, the same method was used for all four groups of hearts. Thus comparisons between the groups of this study appear valid. Future studies will be needed to determine if discrepancies between the results in this study and those from other ischaemia models can be explained by specific CPB-related factors such as moderate hypothermia before inducing ischaemia, global cardiac ischaemia of an empty non-beating heart or other unrecognized factors.

The physiological response induced by β-adrenergic signal transduction in each group was assessed by mechanical studies of isolated trabeculae and papillary muscles. Muscles from the control and open chest groups had comparable mechanical characteristics before and after stimulation with isoprenaline. In general, the mechanical characteristics in the ischaemia and ischaemia–reperfusion groups at baseline, with the exception of time to peak tension, were comparable but significantly depressed as compared to the control and open chest groups. The isoprenaline-mediated mechanical responses in the ischaemia–reperfusion group, except for Vmax, was greater than in the ischaemia group. The contractile response to isoprenaline in the ischaemia–reperfusion group remained lower than in the control and open chest groups, but the magnitude of the tension increase after isoprenaline confirmed that reperfusion allowed a significant recovery of the β-adrenergic responsiveness.

This study represents the first attempt to correlate myocardial function and β-adrenergic signal transduction after CPB. The most striking finding is the discrepancy between the cAMP and tension generation capacities in the ischaemia–reperfusion group. This apparent uncoupling of the effects of adenylate cyclase on the myocardial contractile units suggests that myocardial alterations at a site distal to the production of cAMP occur in the ischaemia–reperfusion hearts, as found in some models of ischaemic [27], hypertrophied [28]and dilated [29]cardiomyopathy. This could explain why stimulation with high extracellular calcium concentration did not reverse myocardial depression in the ischaemia–reperfusion groups. This data interpretation remains speculative because it associates two different parameters in two different preparations. Tension generation reflects a physiological response of a group of intact cells (whole muscle) to β-adrenergic stimulation. In contrast, cAMP generation in a membrane preparation, in presence of a phosphodiesterase inhibitor, reflects the in vitro functional capacity of adenylate cyclase under specific conditions. It does not provide in vivo data about the whole cells where several complex factors other than adenylate cyclase activity may regulate the level of intracellular cAMP. Only an experimental study where direct measurement of intracellular cAMP from intact stimulated muscles could provide an accurate correlation between contractility and cAMP levels.

After stimulation with isoprenaline, the ischaemia–reperfusion muscles and cardiac membranes had absolute responses which resembled the responses in the control and open chest groups rather than the ischaemia group. Thus the increased β-adrenergic receptor density and adenylate cyclase activity in the ischaemia–reperfusion group could represent acute compensatory mechanisms by which the heart attempts to maintain contractility after surgery. Clinical experiences after CPB have shown that the depressed myocardium improves its contractility after β-adrenergic stimulation [30]. This study may illustrate how the heart maintains its responsiveness to β-adrenergic stimulation after cardiac surgery. However, only normal hearts exposed to predefined periods of CPB, myocardial ischaemia and reperfusion were assessed. Further studies will be necessary to determine whether the present results apply in hearts with chronic ischaemic disease or other cardiomyopathy. Moreover the effects of cardioplegia, duration of CPB, internal ventricular defibrillation, duration of global cardiac ischaemia and reperfusion, as well as the time-course of those effects on the β-adrenergic signal transduction pathway during and after CPB remain to be determined in order to better understand the role of myocardial β-adrenergic signal transduction after cardiac surgery.

In summary, this study suggests that the reduction in myocardial contractility that characterizes the ischaemic heart during CPB is accompanied by, and may partially be the result of, a reduction in β-adrenergic signal transduction caused by reduced β-adrenergic receptor density and adenylate cyclase activity. Following reperfusion of the heart and separation from CPB, depression of myocardial contractility is less marked and, in contrast with the findings during ischaemia, is accompanied by increased β-adrenergic signal transduction as determined by the increased cAMP generation in membrane preparations. This appears to be the result of increased β-adrenergic receptor density and adenylate cyclase activity. Decreased levels of Gi{alpha}–2 and G i{alpha}–3 may also contribute to increased cAMP production while uncoupling of the β-adrenergic receptors from the Gs{alpha} protein may serve to temper this effect. However, the increased capacity to generate cAMP in cardiac membranes obtained after CPB is associated with significantly reduced tension generation by corresponding cardiac muscles, as compared to control muscles. These data suggest that cellular alterations distal to cAMP generation rather than changes in β-adrenergic signal transduction may be the primary cause of decreased contractility after CPB. In addition, the changes in β-adrenergic signal transduction observed in this study may explain why the contractile response to β-adrenergic stimulation is poor in the ischaemic heart of cardiac surgical patients while it is usually well preserved in the failing heart after CPB.

Time for primary review 28 days.


   Acknowledgements
 
This study was supported by a grant from the Medical Research Council of Canada. Dr. Anand-Srivastava is a recipient of the Medical Research Council Scientist Award from the Medical Research Council of Canada. Dr. Rouleau is a senior scholar of Les Fonds de la Recherche en Santé du Québec. The authors wish to thank Lise Côté-Dougherty for her excellent technical help, Pierre Lavallée and Yves Thisdale for their expertise as perfusionists and Geraldine Wells for her secretarial assistance and figure preparation.


   Notes
 
1 The results of this study were presented in part at the Annual Meeting of the Canadian Anaesthetists' Society, June 23–27, 1995, in Ottawa, ON, Canada and at the Annual Meeting of the Society of Cardiovascular Anesthesiologists, May 10–14, 1997, in Baltimore, MD, USA. Back

2 Present address: University of Toronto, Cardiology Division, St-Michael's Hospital, 30 Bond Street, Toronto, Ontario, Canada, M5B 1W8 Back

3 Present address: Cell Genesys Inc., 322 Lakeside Drive, Foster City, CA, 94404, USA. Back


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 Abstract
 1 Introduction
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 3 Results
 4 Discussion
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