Journal of Cardiovascular Pharmacology:
Effects of Endogenous Adenosine and Adenosine Receptor Agonists on Hypoxia-Induced Myocardial Stunning in Guinea-Pig Atria and Papillary Muscles
Gardner, Neil M. PhD; Yates, Lisa BSc; Broadley, Kenneth J. DSc
From the Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cathays Park, Cardiff, UK.
Received for publication November 20; 2002; accepted October 30, 2003.
Supported by the Royal Pharmaceutical Society of Great Britain and the British Heart Foundation.
Reprints: Kenneth J. Broadley, DSc, Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cathays Park, Cardiff CF10 3XF, UK. (e-mail: BroadleyKJ@Cardiff.ac.uk).
The effects of endogenous adenosine and adenosine receptor agonists were examined on hypoxia-induced myocardial stunning of guinea-pig isolated paced left atria and papillary muscles. Hypoxia (30 minutes) reduced developed tension and increased diastolic tension (contracture) of left atria (41.8 ± 11.5%) and papillary muscles (17.7 ± 6.2%). Developed tension recovered to 80.8 ± 3.15 and 77.2 ± 5.3% 15 minutes after reoxygenation (stunning). Recovery of left atria was unaffected by adenosine deaminase (1 IU mL−1) but was depressed in papillary muscles (15 minutes, 48.6 ± 4.3%) and contracture (46.1 ± 7.5%) increased. Endogenous adenosine therefore protects from ventricular but not atrial stunning. Adenosine receptor agonists were introduced at 10 minutes into hypoxia. CPA (A1 selective, 3 × 10−8 M) impaired left atrial recovery (5 minutes, 38.1 ± 5.0%), through direct negative inotropy, but did not affect papillary muscles. CGS21680 (A2A selective, 3 × 10−7 M) did not affect recovery. APNEA (A1/A3 receptor agonist, 10−8 M), increased recovery rate of left atria. Improved rate and extent of recovery of papillary muscles by APNEA (15 minutes, 94.8 ± 3.1%) was prevented by the A3 receptor antagonist, MRS-1220 (10−7 M). IB-MECA (A3 selective, 3 × 10−7 M) increased atrial recovery rate but not the maximum developed tension reached in either tissue. However, when added at reoxygenation, IB-MECA caused complete recovery of both tissues, in the absence or presence of adenosine deaminase. Thus, A3 receptor stimulation reverses myocardial stunning of isolated atria and papillary muscles.
Myocardial ischemia and subsequent restoration of normal coronary blood flow causes profound changes in contractile function and electrical activity of the heart. 1,2 Although hypoxia plays a role in the changes produced by ischemia, its effects are compounded by acidosis, lactate accumulation, hyperkalemia and substrate deprivation, which also occur during ischemia. 3 Most studies of electrical and contractile changes of the heart in ischemia and reperfusion have used either whole heart or multicellular preparations of cardiac tissue. 4,5 Although these isolated heart preparations accurately reproduce the physiological situation of ischemia and reperfusion, the interpretation of changes in contractile function is complicated by the coronary vasculature. 6,7 In an attempt to overcome these difficulties, nonperfused isolated cardiac muscle models of ischemia and hypoxia have been developed. 8–10 In these, hypoxia or simulated ischemia cause a decrease in cardiac contractile function. On reoxygenation, cardiac contractility remains impaired—a phenomenon termed myocardial stunning. 11
The degree of stunning can be attenuated by activation of adenosine receptors during ischemia via endogenous adenosine, released during ischemia, 12,13 or through exogenous adenosine added during ischemia. 14 Adenosine receptor activation is also implicated in mediating the protective effect of myocardial preconditioning, 15–17 whereby a brief period of ischemia followed by reperfusion protects the heart from a subsequent more prolonged period of ischemia. 18
Hence, adenosine receptor activation, by adenosine or synthetic analogues, has been shown to be protective in many models of ischemia/reperfusion and preconditioning. In most cases, the cardioprotection has been attributed to activation of A115–17,19,20 or A2A receptors. 21,22 More recently, the A3-adenosine receptor has also been implicated in mediating cardioprotection against experimental infarction or contractile dysfunction in isolated hearts pretreated with an A3 receptor agonist. 23–25 The A3 receptor agonist, IB-MECA, has been shown to attenuate stunning and infarction after coronary artery occlusion in conscious rabbits. 7 Preconditioning has been mimicked by pretreatment of human isolated atrial trabeculae with IB-MECA 26 or rabbit isolated hearts with CB-MECA. 27
The purpose of this study was to establish a model of hypoxia-induced stunning in guinea-pig isolated left atria and papillary muscles and to assess the role, if any, played by endogenous adenosine release in this model. Secondly, the possible cardioprotection against myocardial stunning by exogenously applied adenosine receptor agonists was examined. Since all of the previous cardioprotection studies from other laboratories have used pretreatment with the adenosine agonists, in the present study they are introduced during or at the end of hypoxia. This mode of administration is regarded as being more clinically relevant than pretreatment because myocardial infarction and ischemia are rarely foreseen.
Isolated Atria and Papillary Muscles
Male Dunkin-Hartley guinea-pigs (250–300 g) were killed by a blow to the back of the head followed by exsanguination under running water. The guidelines for the care and use of laboratory animals were followed according to the Animals (Scientific Procedures) Act 1986. The rib cage was opened to expose the heart, and the left atria were removed with cotton threads attached to the tip of the left atrial appendage and the atrioventricular junction. The latter thread attached each atrium to the electrode tips of a bipolar platinum electrode, and the first thread was attached to an isometric transducer (type UF1, 57-g sensitivity range).
After removal of the left atria, a discrete papillary muscle from the left ventricle was exposed by cutting along the interventricular septum. A cotton thread was then placed around the chordae tendineae of the papillary muscle and another through its apical end. The papillary muscle was then dissected from the ventricular wall. The cotton attached around the chordae tendineae was attached to an isometric transducer (type UF1, 57-g sensitivity range) with the cotton through its apical end holding it in close contact with bipolar platinum electrodes. All tissues were set up in 50-mL organ baths containing prewarmed Krebs-bicarbonate buffer gassed with 95% O2/5% CO2 and maintained at 37 ± 0.5°C. Resting diastolic tensions of 0.5–1.0 g and 0.4–0.6 g were then applied to left atria and papillary muscles, respectively.
The tensions developed by both atria and papillary muscles were displayed on an 8-channel Devices MT8P polygraph. Left atria and papillary muscles were electrically stimulated at 2 Hz using square-wave pulses of 5-millisecond duration at threshold voltage + 50%, delivered by a Harvard 50-72 stimulator. The parameters used for electrical stimulation have previously been shown to drive cardiac tissue without causing significant autonomic transmitter releases, when delivered by electrodes in direct contact with the tissue. 28
An equilibration period of 60 minutes was allowed before commencement of an experimental protocol. During this period, the bathing Krebs solution was replaced with fresh Krebs every 15 minutes. The mean basal developed tensions of all groups prior to hypoxia were compared for atria and papillary muscles separately by analysis of variance. The basal developed tensions of neither atria (P = 0.085) nor papillary muscles (P = 0.79) differed significantly between any of the groups (Table 1), although a paired t test showed individual differences between certain groups. Hypoxia was then induced in both left atria and papillary muscles by switching the 5% CO2 in oxygen gassing the bathing solution to 5% CO2 in nitrogen. After the induction of hypoxia, the bathing solution was exchanged several times with fresh CO2/N2 gassed Krebs-bicarbonate solution. The partial pressure of oxygen (pO2) was measured by means of a Clark polargraphic electrode attached to an Instech Laboratories oxygen electrode amplifier (model 102) (Harvard Apparatus Ltd, Edenbridge, Kent, UK). pO2 during hypoxia fell to 46–54 mm Hg compared with 560–620 mm Hg under normoxic conditions. Hypoxia was maintained for 30 minutes before reoxygenation was achieved by returning to gassing with 5% CO2 in oxygen. Tissues were paced throughout.
The effect of adenosine deaminase (AD) was investigated by the addition of 1 IU mL−1 of adenosine deaminase to the bathing medium immediately prior to the induction of hypox-ia. AD remained in the bathing media to the end of each experiment. We have shown that an AD concentration of 0.3 IU mL−1 is sufficient to completely reverse the maximal negative inotropic response of guinea-pig isolated atria to adenosine. 29
Adenosine receptor agonists were added as single concentrations to the tissue bath at 10 minutes into the hypoxic period or at reoxygenation. The concentrations were those found in previous studies to produce between 50 and 100% of the maximal response for the A1 adenosine receptor selective agonist, N6-cyclopentyladenosine (CPA, 3 × 10−8 M), 30 the A2A adenosine receptor selective agonist, CGS21680 (3 × 10−7 M), 31 and the A3 adenosine receptor selective agonist, N6-(3-iodobenzyl)adenosine-5`-N-methyluronamide (IB-MECA, 3 × 10−7 M), 32 or 10-fold greater than the Ki value for binding to the rat A3 receptors for the A1/A3 agonist, N6-2- (-4-aminophenyl) ethyl adenosine (APNEA, 1 × 10−8 M). 33 Agonist or vehicle (polyethylene glycol 400:water 50:50%) remained in the bathing medium to the end of each experiment, unless otherwise indicated.
Drugs and Solutions
The Krebs bicarbonate solution was made up in double distilled water and had the following composition in mM: NaCl 118.0, KCl 4.7, CaCl2.2H2O 2.5, MgSO4.7H2O 1.2, NaHCO3 24.9, KH2PO4.2H2O 1.2, glucose 11.6. All reagents were of Analar grade (Fisons). N6-Cyclopentyladenosine (CPA), 2-[p-(2-carboxyethyl)phenylamino]-5`-N-ethylcarboxamidoadenosine (CGS21680), N6-2-(-4-aminophenyl)ethyladenosine (APNEA) (Sigma, Poole, Dorset, U.K.), and N6-(3-iodobenzyl)adenosine-5`-N-methyluronamide (IB-MECA) (synthesized in-house in the Chemistry Department at GlaxoSmithKline, Renne, France) were all initially dissolved in polyethylene glycol (PEG) 400:water 50:50%. Subsequent dilutions were made in double distilled water. 9-Chloro-2-(-furanyl)-5-[(phenylacetyl) amino][1,2,4]-triazolo[1,5-c]quinazoline (MRS-1220) (Sigma, Poole, Dorset, U.K.) was dissolved in dimethylsulphoxide:PEG:phosphate buffered saline (10:50:40% vol/vol/vol). Adenosine deaminase type VIII from calf intestinal mucosa was purchased from Sigma as a suspension in 3.2 M ammonium sulfate ((NH4)2SO4), pH approximately 6.0.
All results are expressed as group means ± SEM. Comparisons were made between groups at single time points by means of an independent Student's t test. Comparisons of absolute basal developed tensions of atria and papillary muscles for all groups were made by analysis of variance (ANOVA) followed by a post hoc Bonferroni test. Differences were considered significant at a probability value of less than 0.05.
Effects of Hypoxia on Left Atria and Papillary Muscles
A 30-minute period of hypoxia caused a persistent fall in developed tension in both left atria and papillary muscles to 16.9 ± 1.7% and 20.3 ± 1.6% of basal developed tension, respectively (Fig. 1). On reoxygenation, developed tension increased rapidly, the values at 5 minutes post reoxygenation being 77.4 ± 5.0 and 60.2 ± 4.0% in left atria and papillary muscles, respectively, while at 15 minutes, recovery was to 80.8 ± 3.15 and 77.2 ± 5.3% of basal developed tension (Figs. 1 and 2). During the hypoxic period, an increase in basal diastolic tension was observed (Fig. 3). This contracture commenced at 14.0 ± 0.7 and 14.3 ± 1.0 minutes into the hypoxic period in left atria and papillary muscles, respectively, and peaked at 41.8 ± 11.5% and 17.7 ± 6.2% above basal diastolic tension, respectively.
Effect of Adenosine Deaminase
Addition of adenosine deaminase (AD) (1 IU mL−1) caused a transient positive inotropy in atria and papillary muscles (Fig. 1). The peak developed tension in the papillary muscles (144.5 ± 6.5% of preadenosine deaminase basal developed tension, n = 9) was significantly greater (P < 0.05) than in atria (127.8 ± 3.5%, n = 12). The developed tension consistently recovered within 4 minutes. In 2 atria, AD was allowed to remain in contact with the tissue for 30 minutes without exposure to hypoxia. There was an immediate peak increase in tension to 123.9% of basal developed tension, which declined to 114.3, 119.9, and 120.7% at 10, 20, and 30 minutes, respectively. This compared with time-matched control atria (n = 5) in which basal developed tension at 10, 20, and 30 minutes after the equilibration period was 107.8 ± 3.0, 108.4 ± 4.8, and 109.8 ± 7.8%, respectively, of the basal level. It could therefore be assumed that AD would cause no additional decline in basal developed tension during hypoxia. In the presence of AD, recovery of left atria from hypoxia was not significantly affected (Fig. 2A). The developed tensions at 5 and 15 minutes into reoxygenation (69.5 ± 5.5% and 72.0 ± 3.2, respectively) were not significantly (P > 0.05) different from control values. Also, the onset (12.8 ± 0.7 minutes) and peak (47.2 ± 12.6% above resting diastolic tension) of the hypoxic contracture were not significantly (P > 0.05) different from controls (Fig. 3A). In papillary muscles, however, adenosine deaminase had a significant effect on the recovery from hypoxia (Fig. 2B). The developed tension at 5 and 15 minutes (54.4 ± 7.1% and 48.6 ± 4.3, respectively) was significantly less (P < 0.05) than control values. Adenosine deaminase also significantly (P < 0.05) raised the peak increase in diastolic tension (46.1 ± 7.5% above resting) compared with controls (Fig. 3B). Typical experimental traces are shown in Figure 1.
Effects of A1 and A2A Adenosine Receptor Agonists on Recovery from Hypoxia
Addition of a 0.3-mL aliquot of the PEG:water (50:50%) vehicle at 10 minutes into hypoxia and throughout the experiment did not significantly affect the recovery of either left atria or papillary muscles. The developed tensions at 5 and 15 minutes into reoxygenation were 69.4 ± 7.9% and 65.7 ± 7.7% of basal developed tension, respectively, in the left atria and 66.3 ± 4.9% and 80.4 ± 4.5%, respectively, in papillary muscles.
The addition of CPA (3 × 10−8 M) from 10 minutes into hypoxia significantly impaired the recovery of left atria (Fig. 4A). The developed tension at 3 and 5 minutes (27.6 ± 3.5% and 38.1 ± 5.0%) post reoxygenation was significantly less (P < 0.05) than in vehicle controls (50.7 ± 5.4% and 69.4 ± 7.9%, respectively). Since CPA exerts a direct negative inotropy in the left atria, after 5 minutes of recovery the left atria was washed several times to remove the CPA. After this washing, the left atrial developed tension recovered to 66.1 ± 4.4% of basal developed tension at 15 minutes post reoxygenation, which was not different from vehicle controls (65.7 ± 7.7% at 15 minutes post reoxygenation).
CPA (3 × 10−8 M) did not significantly affect the recovery of papillary muscles from hypoxia (Fig. 4B). The developed tensions at 3 and 15 minutes (44.1 ± 8.8% and 74.7 ± 3.2% of basal developed tension, respectively) post reoxygenation was not significantly (P > 0.05) different from the vehicle controls (39.6 ± 5.8% and 80.4 ± 4.4% of basal developed tension, respectively).
CGS21680 (3 × 10−7 M) did not significantly affect the maximum developed tension reached in left atria and papillary muscles on recovery from hypoxia. Developed tensions at 5 and 15 minutes (88.1 ± 6.9% and 82.2 ± 3.5% for left atria and 82.1 ± 6.2% and 90.4 ± 6.1% for papillary muscles, respectively) were not significantly different from vehicle controls (69.4 ± 7.9% and 65.7 ± 7.7% for left atria and 66.3 ± 4.9% and 80.4 ± 4.4% for papillary muscles).
Effects of A3 Adenosine Receptor Agonists on Recovery from Hypoxia
APNEA (10−8 M) caused a significant increase (P < 0.05) in the onset of recovery of left atria from hypoxia. Developed tension at 3 minutes post reoxygenation was 82.7 ± 6.4% compared with 50.7 ± 5.2% for vehicle controls. The developed tensions at 5 and 15 minutes (83.4 ± 5.9% and 77.4 ± 4.3%, respectively), however, were not significantly different from vehicle controls (69.4 ± 7.9 and 65.7 ± 7.7%, respectively) (Fig. 5A). In a second set of experiments, APNEA was washed from the bath at 10 minutes into the regassing. The developed tension at 3 and 5 minutes of reoxygenation (95.3 ± 5.7 and 88.6 ± 4.7%, n = 5) were significantly greater than the vehicle controls, and after washout of the APNEA, there was a small further increase to 103.9 ± 6.7%.
In papillary muscles, APNEA caused a significant improvement in recovery from hypoxia (Fig. 5B). Both the speed and extent of recovery were greater than in controls. The developed tension at 3 minutes post reoxygenation (93.3 ± 3.9%) was significantly greater than vehicle controls (39.6 ± 5.8%). The increases in developed tension at 5 and 15 minutes post reoxygenation (94.8 ± 3.1%, of prehypoxic resting tension) were also significantly greater (P < 0.05) than the vehicle control values (66.3 ± 4.9 and 80.4 ± 4.4%, respectively).
The A3 receptor antagonist, MRS-1220 (10−7 M), added prior to hypoxia in control experiments improved recovery of contractility in both left atria and papillary muscles. At 15 minutes post reoxygenation, atrial and papillary muscle recovery was to 86.9 ± 3.9% and 72.0 ± 8.2%, respectively, compared with the vehicle for MRS-1220 (DMSO:PEG:saline) controls of 65.8 ± 2.7 and 59.3 ± 4.1%, respectively. Thus, in left atria in the presence of MRS-1220 there was little stunning, and it was not possible to evaluate the effect of MRS-1220 on the reversal of stunning by APNEA. In the papillary muscles, however, in the presence of MRS-1220 there was still a substantial degree of stunning (Fig. 6A). When APNEA (10−8 M) was added at 10 minutes into hypoxia in the presence of MRS-1220, there was no improvement in recovery compared with the MRS-1220 control (Fig. 6B). Developed tension at 15 minutes post reoxygenation (51.4 ± 10.0%) was slightly less but not significantly (P > 0.05) different from the MRS-1220 controls (72.0 ± 8.2%) or the vehicle control (59.3 ± 4.1%), but was significantly less (P < 0.05) than with APNEA alone (94.8 ± 3.1%).
IB-MECA (3 × 10−7 M) added at 10 minutes into the hypoxia caused a significant increase in the onset of recovery of left atrial developed tension from hypoxia, the developed tension at 3 minutes post reoxygenation (76.9 ± 7.7%) being significantly greater (P < 0.05) than vehicle controls (50.7 ± 5.4%) (Fig. 7A). The maximum developed tensions reached at 5 and 15 minutes post reoxygenation (86.5 ± 8.2% and 74.0 ± 5.8%, respectively) were not, however, significantly greater (P > 0.05) than vehicle controls (69.4 ± 7.9% and 65.7 ± 7.7%, respectively).
In papillary muscles, IB-MECA (3 × 10−7M) added 10 minutes into hypoxia did not significantly affect (P > 0.05) either the speed or extent of recovery from hypoxia (Fig. 7B). The developed tensions at 3, 5, and 15 minutes post reoxygenation (35.7 ± 5.3%, 74.7 ± 6.3%, and 94.4 ± 9.5%, respectively) were not significantly different from vehicle controls (39.6 ± 5.8%, 66.3 ± 4.9, and 80.4 ± 4.4%, respectively).
When IB-MECA (3 × 10−7 M) was added at reoxygenation in the left atria, recovery of tension was significantly improved (P < 0.05) (Fig. 8A). The developed tensions at 5 and 15 minutes post reoxygenation (96.7 ± 6.5% and 98.5 ± 5.1%) were significantly greater (P < 0.05) than vehicle controls. In papillary muscles, IB-MECA (3 × 10−7 M) added at reoxygenation also caused a significant improvement in recovery (Fig. 8B). The developed tensions at 5 and 15 minutes post reoxygenation (106.7 ± 7.8% and 125.9 ± 3.5%, respectively) were significantly greater (P < 0.05) than vehicle controls (66.3 ± 4.9 and 80.4 ± 4.4%, respectively).
In the presence of adenosine deaminase (1 IU mL−1), IB-MECA (3 × 10−7 M) added at reoxygenation also caused significant improvement (P < 0.05) in recovery from hypoxia of left atria (Fig. 9A). The developed tension at 15 minutes post reoxygenation (87.6 ± 1.5%) was significantly greater than the controls in the presence of AD (72.0 ± 3.2%). In papillary muscles in the presence of AD, IB-MECA (3 × 10−7 M) significantly improved recovery from hypoxia (Fig. 9B). The developed tension at 15 minutes post reoxygenation (84.5 ± 0.7%) was significantly greater (P < 0.05) than the control in the presence of AD (50.5 ± 8.0%).
The contractures were measured as the peak increases in diastolic tension of atria and papillary muscles in the presence of CPA (0.39 ± 0.12 and 0.035 ± 0.01g), CGS21680 (0.10 ± 0.04 and 0.12 ± 0.05 g), APNEA (0.12 ± 0.05 and 0.11 ± 0.03 g) and IB-MECA (0.23 ± 0.07 and 0.19 ± 0.08 g) when added at 10 minutes into the hypoxia. There was no significant difference between these and the corresponding values in the vehicle control (0.22 ± 0.06 and 0.19 ± 0.07 g).
In both the left atria and papillary muscles, hypoxia resulted in a fall in developed tension and an increase in diastolic tension characteristic of contracture. Hypoxia results in an excessive increase in intracellular Ca2+ concentration via the sarcolemmal Na+/Ca2+ exchanger, 34 voltage-dependent slow calcium channels, 35 and through a “leaky” sarcolemma. 36 This calcium overload of the myocardium is thought to be of critical importance in the transition from reversible to irreversible damage in cardiac cells 37 and myocardial contracture, since the application of Na+/H+ exchanger inhibitors 38 and calcium channel antagonist 1,39 are protective against myocardial calcium overload, subsequent infarction, contracture, and stunning. On reoxygenation, there was an incomplete recovery of developed tension in both left atria and papillary muscles, which is known as myocardial stunning and is defined as reversible contractile dysfunction after a period of hypoxia or ischemia. 11,17,40,41 Since electrical activity in the stunned myocardium is normal, 42 the contractile dysfunction probably arises from excitation-contraction uncoupling due to sarcoplasmic reticulum dysfunction, calcium overload, and generation of oxygen-derived free radicals. 43
The isolated cardiac preparations and experimental protocols employed here were designed to investigate myocardial stunning induced by hypoxia rather than by ischemia. Hypoxia is distinct from ischemia 5 which is best defined as low flow hypoxia where the detrimental effects of hypoxia are compounded by oxidative substrate depletion and metabolite pooling. 3 The degree of damage caused by ischemia is probably greater than in this model of hypoxia, where substrate is never depleted and hypoxic metabolites are free to diffuse into the bathing medium. Interpretation of ischemia studies performed in isolated heart preparations or in intact hearts in situ is complicated by the coronary vasculature, which is inherently protective against ischemia, 7 and drugs could mediate myocardial recovery via the coronary vasculature. Thus, even though hypoxic stunning of isolated left atria and papillary muscles is far removed from the physiological situation of ischemia/reperfusion injury, it does provide a useful, uncomplicated tool for studying the underlying mechanism of hypoxia-induced myocardial damage and its modulation by drugs.
Adenosine deaminase terminates the pharmacological actions of adenosine by catalyzing its conversion to inosine. Adenosine released from cardiac myocytes during hypoxia 44 is cardioprotective against ischemia/reperfusion injury. 45 The mechanisms proposed for the cytoprotection by endogenous adenosine include inhibition of Ca2+ influx into cells, 46 prevention of adrenergic neurotransmitters release during ischemia, 47 and increased metabolism through glucose transport. 48 Our results confirm that degradation of endogenous adenosine with adenosine deaminase removes this cardioprotection and enhances the degree of stunning in papillary muscles, although there was no change in the atria. When AD was added to the tissues, there were transient increases in tension, which were greater in papillary muscles. It could be argued that in the presence of AD, a reduced baseline-developed tension during the hypoxia might influence the degree of stunning. However, in control atria, there was no evidence of reduced developed tension over a period of 30 minutes in the presence of AD. Adenosine has also been implicated in delaying the onset of hypoxic/ischemic contracture, which is a measure Ca2+ overload associated with stunning. 39 In the present study, adenosine deaminase, while not affecting the extent of hypoxic contracture in left atria, significantly increased the extent of hypoxic contracture in papillary muscles. Clearly, this indicates that AD prevents the protective actions of endogenously released adenosine in the papillary muscle, presumably by prevention of Ca2+ overload during hypoxia. This confirms its role in the contracture of whole hearts. 39 The lack of effect in isolated atria has not been reported previously but suggests that endogenous adenosine was exerting a protective effect in papillary muscles but not in atria, possibly because levels of adenosine may not be as great in atria. This was supported by the finding that the positive inotropy on adding AD was greater in papillary muscles than in atria. Thus, when endogenous adenosine was removed, the contractures and stunning were equivalent.
Since adenosine receptor activation (by endogenously released adenosine) appears to protect against stunning, the effects of adding selective adenosine receptor agonists at 10 minutes into the hypoxia were next investigated. The selective A1 adenosine receptor agonist, CPA, did not affect recovery from hypoxia in papillary muscles, whereas it slowed recovery of left atria. This slowing of recovery by CPA in the left atria was not due to a worsening of the effects of hypoxia by A1 adenosine receptor activation, but due to the direct negative inotropic effect of A1 adenosine receptor activation in left atria. 29 Hence, when CPA was washed from the bathing medium, recovery of developed tension to the control level was observed. It would have been of interest to determine if a longer recovery period would have allowed further recovery to above control values. CPA did not slow the recovery of the papillary muscle from hypoxia, since A1 adenosine receptor activation does not produce a direct negative inotropy in this preparation. 29
The A2A adenosine receptor agonist, CGS21680, did not affect the degree of stunning in left atria and papillary muscles. Activation of A2A adenosine receptors has been shown to protect against myocardial stunning in isolated working hearts 22 and against myocardial infarction in vivo. 21,49 These effects have been attributed to coronary vasodilatation 22 and inhibition of neutrophil accumulation and superoxide generation. 21 Thus, it was not surprising that no marked effects were observed here in isolated cardiac tissues, since the presence of functional A2A adenosine receptors on myocardial muscle is doubtful. 44
APNEA is an agonist with affinity for both A1 and A3 adenosine receptors. 33 It reduced stunning and quickened recovery in papillary muscles and facilitated a quicker recovery of developed tension in left atria. In a second series of experiments with isolated atria, the APNEA was washed from the bath at 10 minutes into the reoxygenation to determine whether there was an opposing negative inotropy mediated via A1-adenosine receptor activation similar to that seen with CPA. In this group, the developed tension at 10 minutes into reoxygenation had almost recovered to basal level. Thus, there was only a small further recovery on washout of the APNEA. In papillary muscles, no A1 adenosine receptor-mediated direct negative inotropy would be expected with APNEA.
To confirm that A3-receptors were involved in the attenuation of stunning by APNEA, the effect of the A3-adenosine receptor antagonist, MRS-1220, 50 was examined against the APNEA-induced improved recovery. MRS-1220 itself caused some improvement in recovery in both atria and papillary muscles. If endogenous adenosine were protecting the cardiac muscle from stunning through A3 adenosine receptors, MRS-1220 would be expected to worsen the recovery rather than improve recovery. This evidence suggests that A3 adenosine receptors are not activated by endogenous adenosine. Indeed, adenosine itself has relatively low affinity for A3 receptors. 51 The improved recovery in the present study is probably unrelated to blockade of A3 adenosine receptors but may be due to another property of MRS-1220, such as free radical scavenging or blockade of Ca2+ channels, although no reference to such properties was found in the literature. There are claims in the patent literature that A3 receptor antagonists may be cardioprotective against myocardial ischemia and reperfusion injury, 52 although the mechanism is unclear. Only in papillary muscles was there still sufficient stunning in the presence of MRS-1220 for the effect of APNEA to be examined. In this case, APNEA failed to improve recovery in the papillary muscles, and it could be concluded that the effect of APNEA was mediated via A3-adenosine receptors.
IB-MECA was used as a selective agonist of A3 receptors. 53 As with APNEA, there was a more rapid onset of recovery of atrial tension but there was no improvement of papillary muscle recovery from hypoxia. However, when the IB-MECA was introduced at the time of reoxygenation, there was significant recovery of developed tension in both atria and papillary muscles. In papillary muscles, developed tension was in fact raised above the basal value. This, however, cannot be attributed to any positive inotropic activity of IB-MECA, since no such actions have been reported in normoxic cardiac tissues. 30 One possibility is that there is an enhanced overshoot of tension recovery in the presence of IB-MECA. Indeed, this was also evident in the case of IB-MECA added at 10 minutes into hypoxia of isolated atria (Fig. 7A). There was also significant recovery when the experiments were performed in the presence of adenosine deaminase throughout to eliminate any effects of endogenous adenosine. This confirms the results with APNEA that A3 receptor activation can exert a cardioprotective effect against hypoxia-induced myocardial stunning. Previous studies have demonstrated cardioprotection against experimental infarction or contractile dysfunction in isolated hearts pretreated with an A3 receptor agonist. 23–25 Pretreatment with the A3 receptor agonist, IB-MECA, has also been shown to attenuate stunning and infarction after coronary artery occlusion in conscious rabbits. 7 Pretreatment of human isolated atrial trabeculae with IB-MECA 26 or rabbit isolated hearts with CB-MECA 27 has been used to mimic cardioprotection by preconditioning. The present study therefore differs from these earlier studies in that the A3 receptor agonist was administered either during hypoxia or at reoxygenation, which we regard as more clinically relevant than administration prior to the hypoxic/ischemic event. In 1 recent study, the selective A3 receptor agonist, 2-Cl-IB-MECA, was shown to protect against ischemia/reperfusion infarction when administered at reperfusion, but no contractility was monitored. 54 A possible explanation for IB-MECA being more effective in reversing the stunning when administered at reoxygenation is that the A3 receptor undergoes desensitization with prolonged exposure to the agonist. This may occur when IB-MECA is added early in the hypoxic period. In support of this contention, agonist-induced desensitization of recombinant human A3 receptors occurs within 20 minutes and is associated with phosphorylation of the C-terminal by G-protein receptor-coupled kinases. 55
Thus, our results complement earlier studies by showing a direct reversal of contractile dysfunction when A3 adenosine receptor agonists were applied during hypoxia or at reoxygenation, rather than as a preconditioning intervention.
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