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Cardiovascular Research 2006 72(2):292-302; doi:10.1016/j.cardiores.2006.07.020
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Copyright © 2006, European Society of Cardiology

Adenosine A2A receptors are expressed in human atrial myocytes and modulate spontaneous sarcoplasmic reticulum calcium release

Leif Hove-Madsena,*,1, Cristina Prat-Vidala,1, Anna Llacha, Francisco Ciruelab, Vicent Casadób, Carme Lluisb, Antoni Bayes-Genisa, Juan Cincaa and Rafael Francob

aCell Physiology Laboratory, Cardiology Department, Hospital de la Santa Creu i Sant Pau, Institut Catalá de Ciencies Cardiovasculars, Universitat Autònoma de Barcelona, St Antoni Ma Claret 167, 08025 Barcelona, Spain
bDepartment of Biochemistry and Molecular Biology, Universidad de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain

* Corresponding author. Tel.: +34 935565620; fax: +34 935565603. Email address: lhove{at}santpau.es

Received 4 April 2006; revised 25 July 2006; accepted 26 July 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Background: Alterations in the cyclic AMP-dependent regulation of the cardiac ryanodine receptor (RyR2) have been proposed to account for increased spontaneous calcium release from the sarcoplasmic reticulum (SR) in patients with heart failure, ventricular tachyarrhythmias and atrial fibrillation. While the adenosine A2A receptor (A2AR) is known to regulate cyclic AMP levels, expression and function of this receptor in human cardiac myocytes has not been investigated.

Methods: PCR, western blotting and immunofluorescence were used to identify the A2AR, and functional effects of A2AR stimulation were measured with confocal calcium imaging and patch-clamp technique.

Results: The A2AR is expressed in the human right atrium and distributed in a banded pattern along the Z-lines, overlapping with the ryanodine receptor. A2AR stimulation caused a protein kinase A dependent increase in spontaneous SR calcium release in isolated human atrial myocytes. The A2AR agonist CGS21680 increased the frequency of calcium sparks from 0.12±0.03 to 0.31±0.08 sparks·µm min–1 (p<0.05) and calcium waves from 0.65±0.31 to 5.11±1.84 waves·min–1 (p<0.03). Moreover, spontaneous Na–Ca exchange currents (INCX) increased from 1.19±0.17 to 2.50±0.42 min–1 (p<0.001). In contrast, CGS21680 did not alter caffeine inducible calcium release (6.98±0.52 vs. 6.82±0.57 amol pF–1, p=0.6) or the spontaneous INCX amplitude (0.32±0.05 vs. 0.29±0.04 pA pF–1, p=0.2). Current–voltage relationship and amplitude of the L-type calcium current (1.62±0.18 vs. 1.80±0.18 pA pF–1) were not altered, but calcium release dependent inactivation was faster with CGS21680 (13.4±0.7 vs. 15.8±1.0 ms, p<0.001).

Conclusions: Adenosine A2A receptors are expressed in the human atrial myocardium and modulate the frequency of spontaneous calcium release from the SR.

KEYWORDS Adenosine; Arrhythmia (mechanisms); Calcium channel; e–c coupling; SR (function)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Abnormal calcium release from the SR has been associated with heart failure [1–3] and with the genesis of afterdepolarization induced triggered arrhythmias [4–8]. Two main mechanisms have been proposed to explain abnormal calcium release from the SR: mutations and hyperphosphorylation of the SR calcium release channel (ryanodine receptor).

Several mutations in the SR calcium release channel have been shown to increase its opening probability in planar lipid bilayers [5,9,10] and these mutations are found in patients with polymorphic ventricular tachycardia [11–13]. Hyperphosphorylation of the ryanodine receptor mediated by cyclic AMP (cAMP)-dependent protein kinase A (PKA) has been reported in patients with heart failure, exercise-induced ventricular arrhythmias, or atrial fibrillation [2,10,14].

It is therefore conceivable that pharmacological control of receptor mediated cAMP-dependent activation of the ryanodine receptor may be a means to prevent or reduce abnormal calcium release from the SR. In this regard, antagonists of the Gs-protein coupled β-adrenergic receptor have been shown to restore SR calcium release channel function and improve cardiac muscle performance in human heart failure [15]. Theoretically, other Gs-protein coupled receptors, such as the adenosine A2A receptor, may also exert a cAMP-dependent modulation of intracellular calcium handling. Indeed, the A2AR has been shown to cause cAMP dependent inotropic effects in some studies [16–18] and indirect pharmacological data suggest that the A2AR is expressed in rat [16] and guinea pig [19] ventricular myocytes. In contrast, other authors did not find effects of A2AR stimulation [20], and A2AR mRNA was not found in porcine cardiomyocytes [21].

The aim of the present study was to determine whether the A2AR is expressed in the human atrial myocardium and to investigate if this receptor modulates two main players in intracellular calcium handling: the L-type calcium current (ICa) and calcium release from the sarcoplasmic reticulum through the ryanodine receptor. Our results reveal the presence of adenosine A2A receptors in human right atrium and show that agonist stimulation of the A2AR induces a PKA-mediated increase in spontaneous SR calcium release in human atrial myocytes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
2.1. Human samples
Atrial myocardial tissue samples were obtained from 72 patients undergoing cardiac surgery. The average age was 64± 2 years and 60% were male. All patients were in sinus rhythm and subjected to single or combined surgical interventions: coronary artery bypass surgery (28 patients), aortic valve replacement (45 patients), mitral valve replacement (18 patients), other surgical procedures (10 patients). Sixteen patients received angiotensin converting enzyme inhibitors, 12 beta-blockers, 16 diuretics, and 18 statins. Patients treated with calcium channel antagonists were excluded from the study. Specimen was obtained from the right atrial appendage just prior to atrial cannulation for cardioplumonary bypass. Although the atrial tissue samples consisted of tissue that would normally be discarded during surgery, permission to be used in this study was obtained from each patient. The study was approved by the Ethical Committee of our institution and the investigation conforms with the principles outlined in the Declaration of Helsinki. Patients treated with calcium channel antagonists were excluded from the study.

2.2. Myocyte isolation
Myocytes were isolated from human atrial tissue samples as previously described [6]. Briefly, small pieces of tissue were incubated at 35 °C in a Ca2+ free solution containing 0.5 mg/ml collagenase (Worthington type 2, 318 U/mg), and 0.5 mg/ml proteinase (Sigma type XXIV, 11 U/mg solid). After 45 min, the tissue was removed from the enzyme solution, and cells were disgregated in Ca2+-free solution with a Pasteur pipette. The remaining tissue was digested for 3x15 min in a fresh calcium free solution containing 0.4 mg/ml collagenase. Only elongated cells with clear cross striations and without granulation were used for experiments.

2.3. Spontaneous calcium release in isolated myocytes
Calcium sparks and calcium waves were detected in fluo-3 loaded cells using a laser-scanning confocal microscope (Leica TCS SP2 AOBS, Germany) as previously described [6]. The experimental solution contained (in mM): NaCl 136, KCl 4, NaH2PO4 0.33, NaHCO3 4, CaCl2 2, MgCl2 1.6, HEPES 10, Glucose 5, pyruvic acid 5, (pH=7.4). Fluorescence emission was collected between 500 and 650 nm with the excitation at 488 nm attenuated to 1–3%. Calcium sparks and calcium waves were measured at resting conditions. Calcium sparks were detected as an increase in the signal mass of a 3 µm section through the center of a calcium spark, without any detectable increase in an adjacent 3 µm section [6]. An increase in the signal mass in two or more adjacent 3 µm sections were counted as calcium waves. The amplitude of each calcium spark, and its half-life were determined from an exponential fit of the decaying phase of the calcium spark transient. The calcium spark frequency was determined for each cell and normalized to the scanned cell length.

2.4. Patch-clamp
The transient inward Na–Ca exchange current associated with calcium waves was recorded in the perforated patch configuration using a software controlled patch-clamp amplifier (EPC 10, HEKA, Germany). The pipette resistance was 1.5–4 M{Omega}. Experiments were carried out at room temperature and began when the access resistance was stable and had decreased to less than 5 times the pipette resistance. The extracellular solution contained (in mM): NaCl 127, TEA 5, HEPES 10, NaHCO3 4, NaH2PO4 0.33, glucose 10, pyruvic acid 5, CaCl2 2, MgCl2 1.8, (pH=7.4). The pipette solution contained (in mM): aspartic acid 109, CsCl 47, Mg2ATP 3, MgCl2 1, Na2 phosphocreatine 5, Li2GTP 0.42, HEPES 10 and 250 µg/ml amphotericin B, (pH=7.2). The A2AR agonist CGS21680 and the protein kinase A inhibitor H-89 were dissolved in DMSO and kept as 10 mM stock solutions. Ryanodine was kept as a 100 mM stock solution. CGS21680 was used at a final concentration of 200 nm. At this concentration, CGS21680 selectively stimulates the A2AR [22] and induces a robust stimulation of cell shortening in rat ventricular myocytes [16,23]. L-type calcium current amplitude and inactivation were measured at steady state, using repetitive depolarizations from –80 to 0 mV for 200 ms at a frequency of 0.5 Hz.

The caffeine releasable SR calcium content was measured by transiently exposing cells to 10 mM caffeine. The time integral of the resulting Na–Ca exchange current was converted to amoles (10–18 mol) of calcium released from the SR assuming a stoichiometry of 3Na+:1Ca2+ for the Na–Ca exchanger [6].

2.5. Total RNA extraction and PCR
Total RNA was isolated from 15 human right atrial tissue samples using the QuickPrep Total RNA Extraction Kit (Amersham, Freiburg, Germany) according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 µg of total RNA using a random hexamer primer and SuperScript II Rnase H– Reverse Transcriptase (Gibco BRL, Gaithersburg, MD, U.S.A.) according to the manufacturer's protocol. cDNA was amplified using Taq DNA polymerase and the following primers: for the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FGAPDH (5'-GCG GGGCTCTCCAGAACATCAT-3') and RGAPDH (5'-GGT GGTCCAGGGGTCTTACTCC-3') and for the human A2AR (hA2A), FhA2A (5'-GGCTGCCCCTACACATCATCAACT-3') and RhA2A (5'-TGGGCCAGGGGGTCATCT-3').

2.6. Gel electrophoresis and immunoblotting
Membranes from human right atrial tissue or from transiently transfected HEK cells were treated with SDS-PAGE sample buffer (8 M urea, 2% SDS, 100 mM DTT, 375 mM Tris, pH 6.8) by heating at 37 °C during 2 h and resolved by SDS-polyacrylamide gel electrophoresis in 10% gels. Proteins were transferred to PVDF membranes using a semi-dry transfer system and immunoblotted with the indicated antibody and then horseradish-peroxidase (HRP)-conjugated goat anti-rabbit IgG (1/60,000). The immunoreactive bands were developed using a chemiluminescent detection kit (SuperSignal, West Pico Chemiluminescent substrate, Pierce).

2.7. Immunostaining
Human atrial tissue samples used for immunohistochemistry were embedded in OCT and frozen in liquid nitrogen-cooled isopentane. Eight micrometer sections were cut on a cryostat cooled to –18 °C. Sections were collected onto SuperFrost Plus (BDH) slides, air dried and stored at –70 °C. For immunofluorescence, sections were blocked for 30 min in 10% donkey serum in Tris-buffered saline (TBS) (150 mM NaCl/50 mM Tris–HCl, pH 7.5). Slides were incubated with affinity purified anti-A2AR (VC21, 25 µg/ml) for one hour at room temperature and then washed three times for 10 min in TBS. Alexa Fluor® 488 conjugated goat anti-mouse IgG and Texas Red®-conjugated goat anti-rabbit IgG were applied in blocking solution at a dilution of 1:2000. Sections were rinsed and mounted with Vectashield immunofluorescence medium (Vector Laboratories Inc., Burlingame, CA, U.S.A.).

Freshly isolated human cardiac myocytes used for immunocytochemistry were plated in 24 mm glass coverslips coated with poly-D-lysine. After adhesion, cells were rinsed in phosphate-buffered saline and were fixed with 2% paraformaldehyde in PBS for 20 min at room temperature. Cells were washed with PBS and were incubated with 0.1 M glycine for 5 min to quench the aldehyde groups. After washing with PBS, myocytes were permeabilized with 0.2% Triton-X-100 in PBS for 15 min. Cells were then washed with PBS, blocked for 30 min with 10% horse serum at room temperature and incubated with rabbit polyclonal anti-adenosine A2A receptor (10 µg/ml), and either mouse anti-{alpha}-actinin (1:500 dilution), mouse anti-myosin (1:500 dilution), mouse anti-connexin-43 (1:150 dilution), or mouse anti-ryanodine receptor (1:500 dilution) for one hour at room temperature, washed three times with PBS, and stained with anti-mouse Alexa Fluor 488 antibody and anti-rabbit Texas Red. Finally cells were rinsed again for three times and mounted with Vectashield immunofluorescence medium (Vector Laboratories Inc.).

For these studies a Leica TCS 4D confocal laser scanning microscope was used (Leica Lasertechnik GmbH, Heidelberg, Germany).

2.8. Data analysis and statistics
Ca2+ sparks and Ca2+ waves recorded in cells from the same patient were averaged. Unless otherwise stated, average values from each patient were used for statistical analysis and expressed as mean±s.e.m. Data sets were tested for normality. Student's t-test was used to assess significant differences when testing a specific effect. ANOVA was used for comparison of multiple effects and Student–Newman–Keuls post test was used to evaluate the significance of specific effects. Differences were considered statistically significant when p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 References
 
3.1. Expression and localization of A2AR in human right atrium
Fig. 1a and b show that mRNA coding for A2AR was present in right atrium and that both monomeric and dimeric species of the receptor are expressed. The A2AR was expressed in a transversal-banded pattern along the myocardial fiber. A2AR immunostaining was specific since it disappeared when the primary A2AR antibody was omitted or when the A2AR antibody was pre-incubated with the peptide used to raise it (Fig. 1c). The A2AR co-distributed with the cytoskeletal-associated protein {alpha}-actinin at the level of the Z line in the sarcomer and its distribution overlapped with that of the ryanodine receptor (Fig. 2). In contrast, A2AR did not co-localize with the H-band marker myosin or with the gap junction protein connexin-43.


Figure 1
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  		Fig. 1 Expression and localization of A2AR in human right atrium. (a) Human A2AR (hA2AR) and glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) total mRNA expression with hA2AR primers in the absence of any cDNA (negative control, C–), in HUVEC cells (positive control, C+) and in human atrial tissue (Atrium). An experiment representative of six different individuals is shown. (b) Immunoblotting with an anti-A2AR antibody in cell membranes from transiently transfected HEK cells with human A2AR (positive control, C+) and human atrial membranes. (c) Cryosections from human right atrium were stained with anti-A2AR antibody. Immunofluorescent images with and without anti-A2AR antibody are shown in the left and right panels respectively.

 

Figure 2
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  		Fig. 2 Distribution of the A2AR in isolated human atrial myocytes. Isolated human atrial myocytes were stained with anti-A2AR antibody, and with either anti-myosin antibody, anti-{alpha}-actinin antibody, anti-connexin-43, or anti-ryanodine receptor antibody. Superimposed images (merge) and magnification of the area within the white rectangles revealed an overlapping distribution of A2AR with {alpha}-actinin and with the ryanodine receptor (RyR) but not with connexin-43 or myosin. Scale bars, 10 µm (merge) and 5 µm (magnification).

 
3.2. A2AR stimulation and L-type calcium current
To determine if the A2AR modulates L-type calcium current, ICa amplitude and inactivation kinetics were measured before, during, and after exposure of right atrial myocytes to 200 nm of the selective A2AR agonist CGS21680 (Fig. 3a). Fig. 3b shows that a 10 min exposure to CGS21680 had no effect on the ICa amplitude and Fig. 3c reveals that the A2AR agonist significantly shortened the fast, calcium release-dependent, ICa inactivation time constant (tau) from 15.8±1.0 to 13.4±0.7 ms (p<0.001, n=27). Inhibition of calcium release from the SR with 400 µM ryanodine (not shown) increased tau from 13.2±0.9 to 23.5±1.6 ms (n=7, p<0.001) confirming that tau is SR calcium release dependent. Moreover, CGS21680 did not alter tau when added in the presence of ryanodine (23.4±1.4 ms, p=0.92, n=7). Fig. 3d shows the effect of CGS21680 on the current–voltage relationship for ICa.


Figure 3
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  		Fig. 3 Stimulation of the A2AR does not alter L-type calcium current amplitude. (a) L-type calcium currents elicited by a 200 ms depolarization from –80 to 0 mV before (control), during (200 nM CGS), and after (wash) exposure of a human atrial myocyte to CGS21680. (b) Average effect of CGS21680 on ICa amplitude (p=0.2, n=27). (c) Fast time constant for ICa inactivation with CGS21680 (CGS) and without (CON). *** denote p<0.001, n=27. (d) Current–voltage relationship for the ICa amplitude with (CGS) and without (CON) A2AR stimulation.

 
3.3. A2AR activation and spontaneous SR calcium release
To examine if A2AR agonists modulate SR calcium release the effect of CGS21680 on spontaneous SR calcium release in isolated human right atrial myocytes was analyzed. Both local non-propagating calcium release (calcium sparks) and propagating calcium release from the SR (calcium waves) were analyzed. To determine whether A2AR stimulation modifies SR calcium loading or its rate of refilling, we analyzed respectively the amplitude and the decay of 254 calcium sparks from 14 patients. The left panel of Fig. 4a shows a fluo-4 loaded atrial myocyte with the scan line indicated in red. The middle panel shows calcium sparks recorded with this scan line and the right hand panel shows the corresponding changes in the intracellular [Ca2+] for the calcium sparks at the black arrows. Fig. 4b shows that neither the calcium spark amplitude (left panel) nor the decay of the calcium sparks (right panel) were altered by incubation with CGS21680. Fig. 5a shows consecutive line-scan images from a control cell and a cell preincubated with 200 nm CGS21680 and Fig. 5b shows that this A2AR agonist significantly increased both the number of calcium sparks (left panel) and calcium waves (right panel), corresponding to an increase in the calcium wave frequency from 0.65±0.31 to 5.11±1.84 waves min–1.


Figure 4
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  		Fig. 4 Calcium spark amplitude and kinetics are not changed by A2AR stimulation. (a) Bidimensional confocal image of a fluo-3 loaded human atrial myocyte with indication of the scan line used to obtain the line-scan image in the middle panel. The right panel shows the changes in the fluorescence intensity (F/F0) of two calcium sparks occurring at the level of the two black arrows. (b) Agonist stimulation of the A2AR with CGS21680 had no effect on calcium spark amplitude (F/F0) or on the time constant (tau) for the decay of calcium sparks (p=0.36 respectively p=0.08, n=14).

 

Figure 5
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  		Fig. 5 A2AR activation increases spontaneous SR calcium release. (a) Five consecutive line-scan images from a myocyte incubated with the A2AR agonist CGS21680 and without (Control) showing local increases in the intracellular [Ca2+] reflected by a more intense green color. Changes in intracellular calcium in 3 µm sections at the level of the blue and red arrows are shown below the confocal images in the corresponding colors. The scale bars on the left indicate a 1-unit increase in the F/F0 ratio. (b) CGS2160 (CGS) significantly increased the frequency of calcium sparks (left panel) and calcium waves (right panel) in right atrial myocytes from 14 patients. Significant differences between control and CGS21680 are indicated with * (p<0.05) and ** (p<0.01).

 
To determine whether the observed effect of CGS21680 on spontaneous SR calcium release is secondary to an effect on the membrane potential, the patch-clamp technique was used to hold the membrane potential at –80 mV. This voltage clamping did, however, not prevent CGS21680 from reversibly increasing the number of spontaneous calcium waves measured as the Na–Ca exchange current (INCX) activated by them (Fig. 6a). The right hand panel shows that there was no difference in the amplitude of the spontaneous INCX. Fig. 6b shows the time course of the stimulatory effect of CGS21680 and the right hand panel summarizes the stimulatory effect of CGS21680 on INCX (p<0.001, n=27). The effect of CGS21680 was reversed by the selective A2AR antagonist ZM241385 (Fig. 6c). Assessment of the SR calcium loading by rapid exposure of myocytes to 10 mM caffeine at a holding potential of –80 mV gave comparable SR calcium loading before and after agonist activation of the A2AR (7.0±0.5 amol pF–1 and 6.8±0.6 amol pF–1 respectively (p=0.62, n=16, paired t-test).


Figure 6
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  		Fig. 6 Reversibility of A2AR mediated SR calcium release. (a) Spontaneous INCX were recorded with the membrane potential clamped at –80 mV before (control), during (CGS) and after (wash) exposure of a myocardiocyte to 200 nM CGS21680. Vertical scale bars correspond to 100 pA. The right hand panel shows the average INCX amplitude in the presence (CGS) and the absence (CON) of CGS21680 (p=0.16, n=22). (b) Time course of the stimulatory effect of CGS21680 on the number of spontaneous Na–Ca exchange currents (counted as events on each 30 s sweep). The thick line above bars indicates the period of CGS21680 application. The effect of CGS on spontaneous INCX frequency is shown on the right (*** denotes p<0.001, n=27). (c) The A2AR antagonist ZM241385 (1 µM) was able to revert the stimulatory effect of 200 nM CGS21680 on spontaneous INCX. Vertical scale bars correspond to 100 pA. Average values of 5 cells from 3 patients are shown on the right. Spontaneous INCX frequency was significantly higher (p<0.05, asterisk) with CGS-treatment than in control (CON) or with CGS+ZM.

 
Together, these results suggest that the observed effects of CGS21680 are due to a direct effect on the ryanodine receptor. To test this further, the effect of CGS21680 on spontaneous INCX and the caffeine releasable SR calcium content was examined in the presence of tetracaine and ryanodine, two compounds known to inhibit calcium release through the ryanodine receptor. Tetracaine abolished spontaneous INCX and moreover, exposure to CGS21680 in the presence of tetracaine did not induce any spontaneous INCX. Washing of tetracaine induced a transient overshoot in INCX amplitude and frequency (see supplementary figure panel A). This was observed in cells from 5 patients exposed to tetracaine. Application of 400 µM ryanodine to cells from 7 patients irreversibly abolished spontaneous INCX, which did not reappear in the presence of CGS21680. Moreover, ryanodine prevented 10 mM caffeine from releasing calcium from the SR with and without CGS21680, suggesting that caffeine could not overcome the inhibitory effect of ryanodine. In contrast, the time integral of the caffeine induced INCX was increased 3-fold in the presence of 250 µM tetracaine, showing that tetracaine is able to inhibit spontaneous INCX in spite of a 3-fold higher SR calcium load. Simultaneous exposure of the cell to tetracaine and CGS21680 did not further increase the caffeine induced INCX (see supplementary figure panel B and C). These findings further support that A2AR-stimulation increases spontaneous calcium release through the ryanodine receptor.

3.4. Protein kinase A dependency of A2AR-mediated spontaneous calcium release
Since the A2AR is coupled to Gs proteins [24], agonist-mediated receptor activation should increase cAMP levels and this in turn could activate protein kinase A (PKA)-dependent phosphorylation of phospholamban and/or the ryanodine receptor. To test whether A2AR exerts its effect through PKA activation, the selective PKA inhibitor H-89 was employed. As shown in the representative current traces of Fig. 7a, H-89 eliminated the stimulatory effect of CGS21680 in isolated myocytes. Fig. 7b summarizes the average effect of CGS21680 on spontaneous calcium release in the absence and the presence of H-89. In accordance with these results, confocal calcium imaging showed that the spark frequency was significantly lower in myocytes incubated with H-89+CGS21680 than in myocytes incubated with CGS21680 alone (0.018±0.012 versus 0.754±0.260 sparks·µm–1 min–1, p=0.01, n=5). Moreover, spontaneous calcium waves were abolished in myocytes incubated with CGS21680+H-89. These results suggest that the A2AR-dependent stimulation of spontaneous SR calcium release is in fact mediated by PKA activation.


Figure 7
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  		Fig. 7 A2AR mediated SR calcium release is PKA-dependent. (a) Representative current traces recorded before (control) and during application of CGS21680 (CGS) or CGS21680+H-89. (b) Addition of 10 µM H-89 abolished the stimulatory effect of 200 nM CGS21680 in 6 patients. Kruskal–Wallis one way ANOVA on ranks showed a significant effect of the treatment on the wave frequency (p=0.002) and Student–Newman–Keuls post test gave a p<0.05 for CGS+H-89 vs. control (*), for CGS vs. control (**), and for CGS vs. CGS+H-89 (***).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 References
 
This study establishes for the first time that the A2AR is expressed in the human atrial myocardium and demonstrates that this receptor modulates calcium release from the SR in isolated human right atrial myocytes.

4.1. Expression of the A2AR in human right atrium
PCR-technique identified the mRNA of the A2AR and western blotting confirmed the presence of both monomeric and homodimeric forms of the A2AR in the human atrium. Our results confirm previous indirect evidence for the presence of the A2AR in mammalian ventricle [16–19,23]. The striated pattern of the A2AR at the level of the Z-line and its co-distribution with {alpha}-actinin may be explained by a scaffolding action of {alpha}-actinin because {alpha}-actinin interacts directly with the cytoplasmatic C-terminal region of human A2AR and plays a role in the traffic of this receptor [25]. The observed overlap in the distribution of the A2AR and the ryanodine receptor and the positive coupling of A2AR with adenylate cyclase reported in rat ventricular myocytes [18,19], also suggest that an A2AR mediated cAMP-dependent modulation of the ryanodine receptor is feasible.

4.2. Function of the A2AR on L-type calcium current
To address potential functional effects of the A2AR on intracellular calcium handling, we first determined the effect of the A2AR agonist CGS21680 on ICa amplitude. Our results showed no significant effect of CGS21680 on ICa amplitude. A previous study failed to find functional effects of A2AR stimulation in a mammalian ventricular preparation [20] whereas others have shown that A2AR stimulation induces a positive inotropic effect [16–18]. The observation of a positive inotropic effect of CGS21680 in the absence of a concurrent stimulation of ICa as observed here, are not mutually exclusive as long as another calcium source, such as the SR, is activated by CGS21680.

4.3. Function of the A2AR on SR calcium release
In this study we found that A2AR stimulation increases spontaneous calcium release from the SR and reduces the fast time constant for ICa inactivation. Since a reduction in the fast time constant for ICa inactivation is observed with larger SR calcium release [26] our results suggest that A2AR stimulation also triggers calcium induced calcium release from the SR. The stimulatory action of CGS21680 on SR calcium release may occur at the ryanodine receptor or it may be secondary to an effect of CGS21680 on the SR calcium content or on the ICa amplitude, since both of these factors have been shown to regulate SR calcium release [27–30]. Our results suggest that CGS21680 acts on the ryanodine receptor since neither the amplitude of spontaneous INCX, the caffeine releasable SR calcium content, nor the ICa amplitude were altered by CGS21680. Moreover, a stimulatory effect of this compound on SR calcium uptake or SR calcium loading would be expected to speed up the decay and increase the amplitude of calcium spark transients respectively [28,29]. However, CGS21680 elevated the frequency of calcium sparks without changing their amplitude or decay. Finally, the effect of CGS21680 was similar in cells with the resting potential clamped at –80 mV and in unclamped cells, discarding that A2AR mediated effects are secondary to changes in the resting membrane potential, another important modulator of the spontaneous SR calcium release [28,31].

Inositol-triphosphate (IP3) receptors are also expressed in mammalian atrial myocytes and co-localize with the ryanodine receptor [32]. Indeed, a cross-talk between the IP3 receptor and the ryanodine receptor has been shown to modulate spontaneous calcium release from the SR through the ryanodine receptor in mammalian atrial myocytes [33–35]. Theoretically, the stimulatory effect of CGS21680 on spontaneous calcium release could therefore be due to an effect of this compound on IP3-dependent calcium release. However, CGS21680 has been shown to inhibit IP3 production [36]. Moreover, PKA-dependent phosphorylation of the IP3 receptor has been shown to reduce type-3 IP3 dependent calcium release [37] and the type-2 IP3 receptor, which predominates in the heart, is a poor substrate for PKA-dependent phosphorylation [38]. Finally, we found that inhibition of SR calcium release with 400 µM ryanodine or 250 µM tetracaine abolished spontaneous INCX as well as the effects of CGS21680 on spontaneous INCX and fast ICa inactivation, in spite of a three-fold larger SR calcium load with 250 µM tetracaine. Taken together, this suggests that activation of the A2AR with CGS21680 stimulates the ryanodine receptor itself.

4.4. Protein kinase A dependent activation of SR calcium release
The A2AR is positively coupled to adenyate cyclase through Gs proteins and their stimulation has been reported to increase cAMP levels in rat ventricular myocytes [18,19]. Stimulation of SR calcium release by the A2AR agonist CGS21680 could therefore result from a PKA-dependent stimulation of the ryanodine receptor in a manner similar to that reported for Gs protein-coupled β-adrenergic receptors [3,15] in patients with failing hearts. In these patients, a PKA-dependent phosphorylation of the ryanodine receptor has been proposed to cause an increased calcium leak from the SR [2,15]. While this proposal is controversial [39,40] our finding that H-89 abolished the stimulatory effect of CGS21680 on spontaneous SR calcium release does confirm a positive coupling between the A2AR, PKA, and the ryanodine receptor in human atrial myocytes. Moreover, our findings suggest that the A2AR mediates a PKA-dependent regulation of the ryanodine receptor itself, since A2AR stimulation had no effect on ICa amplitude, spontaneous INCX amplitude, decay of calcium sparks, or the amount of calcium that could be released from the SR by a rapid caffeine application.

The lack of effect of CGS21680 on SR calcium loading and calcium spark decay is also in accordance with a study showing that A2AR stimulation in guinea pig ventricle increases the cellular cAMP content but not phospholamban phosphorylation [19]. Furthermore, there is growing evidence that cAMP-mediated pathways are locally controlled [41–43] and a local A2AR-mediated control of cAMP could explain why CGS21680 stimulates SR calcium release without affecting SR calcium loading, calcium spark decay, or ICa amplitude.

In view of the reported PKA-dependent hyperphosphorylation of the ryanodine receptor in heart failure [2,3,15] and in catecholaminergic polymorphic ventricular tachycardia [4], it will be important to determine whether the A2AR is expressed and modulates spontaneous SR calcium release in the diseased ventricular myocardium.

In summary, we show that the adenosine A2A receptor is expressed in the human atrial myocardium and has the potential to modulate SR calcium release in human atrial myocytes. Therefore, pharmacological control of A2AR activation may offer a novel approach to control calcium release from the SR.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.07.020.


   Acknowledgements
 
This work was supported by Ramon y Cajal Grants to L. Hove-Madsen and F. Ciruela, and grants from the Spanish Ministry of Science and Technology (SAF2001-3474), Red de investigación cooperativa de las enfermedades cardiovasculares del Instituto de Salud Carlos III (C03/01), Fundació Mutua Universal, and Sociedad Española de Cardiología.


   Notes
 
1 These authors contributed equally to this work. Back

Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 

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