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Cardiovascular Research 2003 60(3):529-537; doi:10.1016/j.cardiores.2003.09.012
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Altered signal transduction in cardiac ventricle overexpressing A1-adenosine receptors

Joachim Neumanna, Peter Boknik*,a, Frank Begrowa, Gabriela Hanskea, Isabel Justusa, Marek Mat'usa, Uta Reinkea, G.Paul Matherneb and Wilhelm Schmitza

aInstitut für Pharmakologie und Toxikologie, Universitätsklinikum Münster, Westfälische Wilhelms-Universität, Domagkstraße 12, D-48129 Münster, Germany
bDepartment of Pediatrics, University of Virginia, Charlottesville, VA, USA

*Corresponding author. Tel.: +49-251-8355510; fax: +49-251-8355501. Email address: boknik{at}uni-muenster.de

Received 8 February 2003; accepted 3 September 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: The aim of the present study was to assess the effects of A1-adenosine receptor (A1-AR) stimulation in ventricle of A1-adenosine receptor overexpressing mice (transgenic mice, TG). Methods: Effects of the A1-adenosine receptor agonist R-PIA ((–)-N6-phenylisopropyladenosine) on phosphorylation of phospholamban (PLB), Ca2+ transients, Ca2+ currents and cell shortening were studied in isolated ventricular cardiomyocytes. Results: R-PIA alone did not affect contractility in isolated electrically stimulated cardiomyocytes from wild-type mice (WT) or TG. However, after pre-stimulation of β-adrenoceptors by isoproterenol, R-PIA reduced contractility in cardiomyocytes from WT but increased contractility in TG. Under the same conditions, R-PIA reduced isoproterenol-stimulated currents through L-type Ca2+ channels, Ca2+ transients and phosphorylation of PLB in cardiomyocytes from WT. In contrast, R-PIA diminished phospholamban phosphorylation induced by isoproterenol but augmented isoproterenol-elevated currents through L-type Ca2+ channels, and isoproterenol-heightened Ca2+ transients in cardiomyocytes from TG. Conclusions: We suggest that A1-adenosine receptor overexpression reverses the interaction of β-adrenergic and A1-adenosine receptor stimulation, at least in part. Hence, the receptor/effector coupling is dependent on receptor density in this model.

KEYWORDS Adenosine; Blood pressure; Ca-channel; Contractile function; Protein phosphorylation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Adenosine exerts negative inotropic, negative dromotropic and negative chronotropic effects in the heart [1]. These effects can occur in the presence of adenosine alone (direct effect) and/or in the presence of β-adrenergic stimulation (indirect effect). Adenosine can act on adenosine receptors on the outside of the sarcolemma. These consist of A1-, A2a-, A2b- and A3-adenosine receptors [2–4]. A1-adenosine receptors (A1-AR) mediate the (direct and indirect) negative inotropic, chronotropic and dromotropic effects of adenosine in the heart [5]. A1-AR stimulation can increase an inwardly rectifying potassium channel current (IK-Ado). This current is present in the atrium but small or absent in the ventricle (with the exception of rat or ferret). A1-AR stimulation per se has no or a very small effect on current through L-type Ca2+ channels. Likewise, adenosine alone poorly inhibits adenylyl cyclase activity. However, adenosine profoundly reduces the β-adrenoceptor stimulated L-type Ca2+ current [5]. Moreover, adenosine affects via A1-AR β-adrenoceptor-mediated phosphorylation of phospholamban (PLB), a key regulator of sarcoplasmic Ca2+-ATPase (SERCA). SERCA pumps Ca2+ from the cytosol into the sarcoplasmic reticulum. In this way, relaxation occurs faster and more Ca2+ is available for the next heart beat. The affinity of SERCA for Ca2+ is reduced by dephosphorylated PLB. Once PLB is phosphorylated, for instance by cAMP dependent protein kinase (PKA) at serine 16, the transport rate of SERCA at low Ca2+ is stimulated. β-Adrenergic stimulation, e.g. by isoproterenol, increases cAMP content and cAMP activates PKA. The subsequent phosphorylation of PLB and disinhibition of SERCA explains, at least in part, the positive inotropic and relaxant effect of β-adrenergic stimulation [6]. A1-AR stimulation decreases the phosphorylation of PLB stimulated by isoproterenol in ventricular cardiomyocytes from rat and guinea pig [7–9].

Mice that overexpress the A1-adenosine receptor under control of the {alpha}-myosin heavy chain promoter selectively in the heart have been generated [10] (unless stated otherwise, these mice are abbreviated transgenic mice). A1-adenosine receptor overexpression in these mice causes bradycardia in intact animals and in isolated perfused hearts [10,11]. Moreover, the mice were significantly more resistant to both the functional and metabolic effects of ischaemia [10,12].

Our initial characterization of contractility in isolated atrial preparations from these transgenic mice detected a—paradoxical—positive inotropic effect of adenosine alone or after β-adrenergic stimulation. These positive inotropic effects of adenosine in atrium were A1-adenosine receptor mediated. The negative chronotropic effect of adenosine was, however, maintained in these transgenic mice [13]. No experiments to assess the inotropic effects of adenosine and their signal transduction in the ventricle of these transgenic mice have yet been reported.

Hence, we addressed the following questions: (i) how does A1-adenosine receptor stimulation affect contractility in cardiomyocytes from transgenic animals when given alone; (ii) how does A1-adenosine receptor stimulation affect contractility in cardiomyocytes from transgenic animals when given after β-adrenergic stimulation; and (iii) what signal transduction mechanism(s) mediate(s) these effects.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animals
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The construct design and initial characterization of the transgenic lines has been previously reported [10]. The rat A1 adenosine cDNA was expressed under the control of the modified {alpha}-myosin heavy chain (MHC) promoter. Three transgenic lines, 1, 5 and 8, were established. In ligand-binding experiments, their Bmax for A1-adenosine receptors was about 2600, 3300 and 250 fmol mg–1 protein, respectively, whereas levels in wild-type mice (WT) were around 8 fmol mg–1 protein. In the present study, only the line 1 was investigated. Identification of transgenic mice was performed by PCR in genomic DNA isolated from tail biopsies as described [13].

2.2. Measurement of blood pressure and heart rate
The systolic, mean, and diastolic blood pressures were measured in conscious mice with the tail-cuff method [14] using a Sphygmomanometer (BP-98A, Softron, Tokyo, Japan) and recorded on a monitor. Heart rate was computed from the amplitude of the pulse signal. The mice were held in the chamber for 10 min before the measurements were started. Twenty-five single measurements made over a period of 5 days were averaged for each mouse.

2.3. Surface electrocardiogram measurement
Surface resting electrocardiograms were performed in wild-type and transgenic mice anesthetized with 15 ml/kg of 2.5% Avertin® (2,2,2-tribromoethanol) via i. p. injection. After the induction of anesthesia, the mice were positioned on a 37 °C heating pad and a three-lead electrocardiogram was obtained by placing subcutaneous needle electrodes in the limbs. The signal was amplified with a ECG Amplifier (Föhr Medical Instruments MVV-0608, Seeheim/Ober-Beerbach, Germany) and digitized at 10.0 kHz with a PowerLab recording unit and Charts 4.0 Windows (both from AD Instruments, Grand Junction, CO, USA). For each animal heart rate, RR, PQ, QRS and QT intervals were measured in 10 beats from both lead I and II and averaged.

2.4. Gel electrophoresis and Western blotting
Gel electrophoresis and Western blotting were performed as described [15]. The A1-adenosine receptor protein was quantified using the polyclonal antibody raised against a synthetic polypeptide corresponding to carboxy-terminal domain (amino acids 309–326) of the rat A1-adenosine receptor (Affinity Bioreagents, Golden, CO, USA). Protein expression of Gi{alpha}-subunit was measured by means of polyclonal antibody against the carboxy-terminal peptide CKNNLKDCCLF of Gi{alpha} (Calbiochem, San Diego, CA, USA). Both primary antibodies were detected using [125I]-labeled protein A and visualized and quantified in a PhosphorImagerTM using ImageQuaNTTM software (Molecular Dynamics, Sunnyvale, CA, USA). Western blotting for calsequestrin, phospholamban and sarcoplasmic Ca2+-ATPase was performed as described previously [16].

2.5. Isolation of cardiomyocytes
Ventricular cardiomyocytes were isolated from wild-type and A1-adenosine receptor overexpressing mouse hearts using a published protocol [17]. Animals were pretreated with heparin (5 U/g body weight), and later anesthetized with CO2. Mouse hearts were excised and the cannulated aorta was fixed to a Langendorff apparatus. Hearts were perfused for 5 min at 2 ml/min with a Ca2+-free solution (solution A) composed of (in mM) 140 NaCl, 5.8 KCl, 0.5 KH2PO4, 0.4 NaH2PO4, 0.9 MgSO4, 10 HEPES, 11.1 glucose (pH 7.1), followed by a perfusion for 30 min with solution A supplemented with 0.2 mg/ml collagenase (type D, Boehringer Mannheim, Mannheim, Germany). Ca2+ concentration was gradually increased during digestion to 100 µM. After enzymatic digestion, the hearts were perfused for 10 min with solution A. The ventricles were cut into several pieces and subjected to gentle agitation through a nylon mesh to separate the cardiomyocytes. All subsequent experiments on isolated cardiomyocytes were performed in the presence of adenosine deaminase (10 U/ml) to avoid interference from endogenous adenosine on treatment [8].

2.6. Measurement of phospholamban phosphorylation
The drug solution (100 µl) was preincubated at 37 °C for 2 min before mixing with 100 µl of the diluted cardiomyocytes and kept at 37 °C. The reaction was stopped by adding 100 µl of SDS stop solution [18]. Samples were resolved by polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The phosphorylation of phospholamban was assessed using a polyclonal antibody directed against the PLB-peptide (residues 9–19) phosphorylated at serine –16 (PhosphoProtein Research, Bardsey, UK) [19] as recently described [20].

2.7. Cardiomyocyte shortening measurements
Contractility of isolated cardiomyocytes was recorded with a Panasonic video recorder AG-7350 (Matsushita Electric Ind., Japan). Cardiomyocyte shortening was visualized on a monitor (Pieper WV-5410, Pieper, Mellingen, Switzerland), and connected to a video integrator (Pieper Video Integrator 310A) interfaced to a video camera (Pieper Kamera FK7512-IQ). The video camera was attached to a Leica microscope (Wetzlar, Germany). Edge detection measurements were performed at room temperature on intact, rod-shaped cells, which had no spontaneous contractions or microblebs. Cells were placed in a chamber (1 ml), and perfused at 0.5–1 ml/min with solution A containing 1 mM CaCl2. Cardiomyocytes were stimulated at 0.5 Hz with platinum electrodes placed on the sides of the experimental chamber. The maximal magnitude of contraction was normalized to resting cell length and expressed as percent of shortening. The basal shortening did not differ between WT and TG cardiomyocytes and amounted to 2.08±0.25 µm (21 cells from 13 animals) in WT and to 2.02±0.19 µm (27 cells from 15 animals) in TG, respectively.

2.8 Whole-cell L-type Ca2+ current
Single cardiomyocytes were studied using the whole-cell variation of the patch-clamp technique. Recordings were performed under conditions that suppress Na+ and K+ currents [21]. Briefly, cells were plated in a small dish (2 ml) on the stage of an inverted microscope (Leica, Cologne, Germany). The extracellular solution was composed of (in mM): TEA-Cl 130, MgCl2 1, 4-aminopyridine 4, HEPES 10, dextrose 10, CaCl2 2, adjusted to pH 7.3 with TEA-OH. The micropipette electrodes (resistances 1.5–2.5 M {Omega}) were filled with (in mM): K-aspartate 80, KCl 50, KH2PO4 10, MgCl2 0.5, MgATP 3, HEPES 10, EGTA 1, adjusted to pH 7.4 with KOH. All experiments were done at room temperature. Currents were elicited by voltage steps from a holding potential of –40 mV to a test potential of +10 mV for 200 ms, applied every 10 s. Cell capacitance and ICa were recorded with an L/M-PC amplifier (LIST-Electronic, Darmstadt, Germany) according to standard protocols. Data were computed with the ISO2 software (MFK, Niedernhausen, Germany). The basal Ca2+ currents were comparable and amounted to 5.54±0.33 pA/pF (30 cells from 12 animals) in WT and to 5.12±0.27 pA/pF (47 cells from 17 animals) in TG cardiomyocytes, respectively.

2.9 Measurement of Ca2+ transients
Cardiomyocytes were incubated for 5 min at room temperature with solution B containing (mM) in 140 NaCl, 5.8 KCl, 0.5 KH2PO4, 0.4 NaH2PO4, 0.9 MgSO4, 10 HEPES, 11.1 glucose, 2.5 CaCl2 and 50 mg/l ascorbic acid supplemented with 25 µM Indo-1/AM. The cells were then superfused with solution B without dye for 45 min before the measurements started. Indo-1 fluorescence was recorded at room temperature from single myocytes using a dual-emission microfluorescence system (Photon Technologies, South Brunswick, NJ). Excitation was at 365 nm, and the emitted fluorescence was recorded at 405 and 495 nm. The ratio of fluorescence at the two wavelengths was used as an index of cytosolic Ca2+ concentration. Cardiomyocytes were stimulated at 0.5 Hz with platinum electrodes placed on the sides of the experimental chamber. Data were collected at 20 Hz, and acquisition and processing were supported by Felix 1.1 software (Photon Technologies). Basal Ca2+ transients were comparable in TG and WT and the difference of ratio fluoroscence between systole and diastole amounted to 0.11±0.005 (21 cells from 13 animals) in WT and to 0.11±0.005 µm (27 cells from 15 animals) in TG, respectively.

2.10. Chemicals
The following compounds were used: adenosine deaminase, isoproterenol (Boehringer Mannheim), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino] ethyl]benzenepropanoic acid hydrochloride (CGS21680C, both from Tocris Cookson, Ellisville, MO, USA), Indo-1/AM (Molecular Probes, Eugene, OR, USA), (–)-N6-phenylisopropyladenosine (R-PIA, Sigma, St. Louis, MO, USA). R-PIA, DPCPX as well as CGS21680C were solubilized in DMSO. The stock solutions in DMSO (0.02 mM) were further diluted in appropriate aqueous buffers for measurements. The final concentration of DMSO in all protocols was set to 0.1%. This concentration of DMSO alone has no effect on Ca2+ currents in wild-type and transgenic cardiomyocytes (data not shown). The other chemicals were of best analytical grade. Twice distilled water was used throughout.

2.11. Statistics
Results are expressed as mean±S.E.M. Significance was estimated by Student's t-test for paired or unpaired observations, as appropriate. A P-value lower than 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Blood pressure and echocardiography
First, we wanted to gain further insight into the physiological consequences of A1-adenosine receptor overexpression in intact animals. Systolic blood pressure, diastolic blood pressure and mean blood pressure were not changed in conscious transgenic mice as compared to wild-type mice (Table 1). However, overexpression of A1-adenosine receptors led to reduced heart rate in the intact, nonsedated mouse (664.3±18.5 bpm in WT vs. 590.7±16.1 bpm in TG, Table 1). Bradycardia was also noted using another independent method, i.e. surface ECG. The RR interval (inversely related to the beating rate) was larger in transgenic mice which indicated a lower beating rate (133.3±2.9 ms in WT vs. 157.0±8.2 ms in TG, Table 2). Of note, the beating rates obtained from ECG were lower than the corresponding values from blood pressure measurements, probably due to the inhibitory effect of anaesthesia. The PQ (36.0±1.2 ms in WT vs. 48.0±2.3 ms in TG) and QRS (28.7±0.7 ms in WT, 36.7±0.7 ms in WT) intervals were prolonged which implies lower conduction rates from the SA node to the AV node (PQ) and in the ventricle (depolarization, QRS).


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  		Table 1 Blood pressure and heart rate in conscious wild-type and A1-adenosine receptor overexpressing (A1-AR) mice

 

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  		Table 2 ECG parameters in anesthesized wild-type and A1-adenosine receptor overexpressing (A1-AR) mice

 
3.2 Expression of cardiac regulatory proteins
Next, we assessed the expression of gene products possibly relevant for signal transduction in this model based on the literature. We characterized the ventricular myocardium with regard to A1-adenosine receptor protein overexpression, Ca2+-regulatory proteins and pertussis toxin-sensitive Gi-proteins. As seen in Fig. 1, we could detect A1-adenosine receptors using antibodies in samples from transgenic ventricles. However, no specific signal using the anti-A1-adenosine receptor antibody was measurable in wild-type ventricular preparations. The negative inotropic effects of adenosine are mediated by pertussis toxin-sensitive Gi-proteins [23,24]. However, their expression was similar in wild-type and transgenic mice (Table 3). Likewise, the expression of phospholamban (PLB), sarcoplasmic Ca2+-ATPase (SERCA) as well as calsequestrin (CSQ) was comparable in wild-type and transgenic ventricular samples (Fig. 1; Table 3).


Figure 1
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  		Fig. 1 Autoradiogram of nitrocellulose strips. Protein expression of sarcoplasmic Ca2+-ATPase (SERCA), calsequestrin (CSQ), {alpha}-subunit of inhibitory G-protein (Gi{alpha}), A1-adenosine receptor (A1-AR) and phospholamban (PLB) in ventricles of wild-type (WT) and A1-adenosine overexpressing mice (TG). Immunological quantification was performed as described in Methods. Radioactive bands were visualized and quantified using a PhosphorImagerTM. On the left, molecular weight markers are indicated. Protein expression of Gi{alpha}, PLB, CSQ and SERCA did not differ between wild-type and A1-adenosine overexpressing animals.

 

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  		Table 3 Protein expression in ventricles of wild-type and A1-adenosine receptor overexpressing (A1-AR) mice

 
3.3. Phospholamban phosphorylation
A reduction of isoproterenol-stimulated phospholamban phosphorylation was here noted in wild-type mouse cardiomyocytes. Isolated mouse ventricular cardiomyocytes were stimulated with 10 nM isoproterenol alone or by additionally applied (–)-N6-phenylisopropyladenosine (R-PIA, 10 µM), an A1-adenosine receptor agonist. Isoproterenol elevated phosphorylation of PLB which was immunologically detected using a phosphorylation-specific antibody. This phosphorylation was attenuated by R-PIA from 147.3±7.9% to 122.7±3.5% of control in transgenic and from 148.1±5.9 to 129.2±3.9% of control, respectively, in wild-type cardiomyocytes (Fig. 2).


Figure 2
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  		Fig. 2 Phosphorylation of phospholamban (PLB) at serine-16. Ventricular cardiomyocytes from wild-type (WT) and A1-adenosine receptor overexpressing (A1-AR) mice were stimulated with isoproterenol (10 nM, Iso) alone, or with Iso plus (–)-N6-phenylisopropyladenosine (R-PIA, 10 µM) or with solvent control (Ctr). Samples were subjected to gel electrophoresis, transferred to nitrocellulose and incubated with an antibody specific for PLB phosphorylated on serine 16 and a radioactive secondary antibody for visualization. The ordinate gives change in phosphorylation as percent of Ctr. Crosses denote a significant difference versus Iso alone (p<0.05). Effects of R-PIA on Iso-stimulated phosphorylation of PLB were comparable in wild-type and A1-AR-overexpressing animals.

 
3.4. Cell shortening
R-PIA (0.1–10 µM) alone had no direct effect on cell shortening in wild-type or transgenic cardiomyocytes (data not shown). In wild-type cardiomyocytes, 10 µM R-PIA diminished isoproterenol (10 nM)-stimulated contractility to 72.4±6.0% of isoproterenol value. However, R-PIA increased isoproterenol (10 nM)-stimulated contractility even further to 132.9±11.4% of isoproterenol value in transgenic cells (Fig. 3). In order to study the underlying mechanism of this A1-AR-mediated increase of contractility, measurements of Ca2+ currents and Ca2+ transients were performed.


Figure 3
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  		Fig. 3 Cell shortening. Ventricular cardiomyocytes isolated from wild-type (WT) and A1-adenosine overexpressing (A1-AR) mice were electrically stimulated and contractility was assessed. Cardiomyocytes were stimulated by isoproterenol (10 nM, Iso) alone, or with Iso plus (–)-N6-phenylisopropyladenosine (R-PIA, 10 µM) or with solvent control (Ctr). The ordinate gives change in cell contractility as percent of the effect of Iso. Iso increased contractility to 159±17% of Ctr in WT and to 166±19% of Ctr in A1-AR cardiomyocytes. Asterisk denotes a significant difference versus wild-type animals (p<0.05), crosses denote a significant difference from Iso alone (p<0.05). The effect of R-PIA on contractility was reversed in A1-AR-overexpressing cardiomyocytes.

 
3.5 Ca2+ currents
Ca2+ currents through L-type Ca2+ channels were measured using the patch-clamp technique in the whole cell configuration. R-PIA (0.1–10 µM) alone did not affect Ca2+ currents, neither in wild-type nor in transgenic cardiomyocytes (data not shown). Isoproterenol (10 nM) amplified the Ca2+ current in both wild-type and transgenic cells. Additionally applied 10 µM R-PIA decreased the current to 80.9±3.5% of isoproterenol value in wild-type cardiomyocytes. In contrast, this concentration of R-PIA further increased isoproterenol-stimulated Ca2+ currents to 115.7±2.0% of isoproterenol value in transgenic cardiomyocytes (Fig. 4). On the other hand, R-PIA led to a slight decrease of isoproterenol-stimulated Ca2+ currents in a lower concentration range (0.1 and 1 µM) in transgenic cardiomyocytes. All effects of R-PIA in wild-type as well as in transgenic cardiomyocytes could be completely blocked by the selective A1-adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 µM), indicating A1-AR-mediated effects (data not shown). In order to address the possible involvement of A2-AR stimulation, we studied effects of the A2-AR-agonist CGS21680C. CGS21680C (0.01–1 µM) alone did not affect Ca2+ currents in wild-type or transgenic cardiomyocytes (data not shown). Similarly, CGS21680C had no effect on the isoproterenol-stimulated Ca2+ currents in transgenic cardiomyocytes. However, a slight decrease of isoproterenol-stimulated Ca2+ currents by 1 µM CGS21680C was observed in wild-type cardiomyocytes (from 193.9±9.4% to 172.3±10.1% of control, n = 4 each, p<0.05).


Figure 4
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  		Fig. 4 Currents through L-type Ca2+-channels. (A) Ventricular cardiomyocytes from wild-type (WT) and A1-adenosine receptor overexpressing (A1-AR) mice were patch-clamped and stimulated with isoproterenol (10 nM, Iso) alone, or with Iso plus (–)-N6-phenylisopropyladenosine (R-PIA, 10 µM) or with solvent control (Ctr) as indicated. The ordinate bar gives current in pA. The abscissa bar gives the time in ms. (B) The ordinate gives change in current as percent of the effect of Iso. Iso increased the Ca2+ current to 194±14% of Ctr in WT and to 204±15% of Ctr in A1-AR cardiomyocytes. Asterisk denotes a significant difference versus wild-type animals (p<0.05), crosses denote a significant difference from Iso alone (p<0.05). In contrast to WT, R-PIA at 10 µM increased Iso-stimulated Ca2+ currents in A1-AR-overexpressing cardiomyocytes.

 
3.6 Ca2+ transients
Similar results were obtained for Ca2+ transients studied by means of the Indo-1 method in cardiomyocytes. Ca2+ transients were incremented by isoproterenol in wild-type and transgenic cells (Fig. 5). While R-PIA depressed isoproterenol-induced Ca2+ transients to 88.3±2.0% in wild-type cells, it even augmented isoproterenol-stimulated Ca2+ transients to 120.3±4.0% in transgenic cells. R-PIA (0.1–10 µM) alone did not affect the parameters listed above (data not shown).


Figure 5
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  		Fig. 5 Ca2+ transients. Ventricular cardiomyocytes from wild-type (WT) and A1-adenosine receptor overexpressing (TG) mice were loaded with Indo-1, electrically stimulated and incubated with isoproterenol (10 nM, Iso) alone, or with Iso plus (–)-N6-phenylisopropyladenosine (R-PIA, 10 µM) or with solvent control (Ctr). The ordinate gives change in alteration of Ca2+ transients as percentage of the effect of Iso. Iso increased the Ca2+ transients to 248±7% of Ctr in WT and to 226±18% of Ctr in TG cardiomyocytes. Asterisk denotes a significant difference versus wild-type animals (p<0.05), crosses denote a significant difference from Iso alone (p<0.05). The effect of R-PIA on Ca2+ transients was reversed in A1-AR-overexpressing cardiomyocytes.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The main new finding of the present study was the following: A1-adenosine receptor (A1-AR) stimulation in the presence of β-adrenergic activation led to a paradoxical increase of contractility in ventricular cardiomyocytes of a transgenic mouse line overexpressing A1-adenosine receptors.

4.1. Inotropic considerations
There are regional and species differences of the A1-AR/effector coupling in the heart. In atrium, A1-AR stimulation exerts a direct negative inotropic effect. The main mechanism of this effect involves opening of potassium channels which results in shortening of action potential duration. [23,24]. The mechanism(s) of A1-adenosine action is(are) more complex in the ventricle. At least in guinea pig and mouse, A1-AR stimulation alone does not reduce contractility [25]. This is explained by a lack of expression of potassium channels that couple to adenosine receptors. In ferret and rat, the appropriate potassium channel seems to couple as A1-AR-agonists alone decreases contractility in ventricular cardiomyocytes [25]. In all species mentioned above, A1-AR activation led to a negative inotropic effect in the presence of cAMP-increasing agents. This is sometimes called the indirect or anti-adrenergic effect of adenosine [4].

Previous studies raised the question how adenosine receptor overexpression might change the effects of adenosine in the ventricle. Stimulation of A1-adenosine receptors by agonists like R-PIA alone did not show any inotropic effect in wild-type [25] or transgenic ventricular mouse cardiomyocytes (this report). Hence, in transgenic ventricle like in wild-type ventricle, A1-adenosine receptors may not activate potassium channels in mouse (like in guinea pig). However, after β-adrenergic stimulation, R-PIA elevated contractility further in isolated ventricular cells of transgenic mice. In wild-type cardiomyocytes contractility was decreased. The effects of R-PIA in wild-type as well as in transgenic cardiomyocytes were completely abolished by DPCPX and therefore mediated by A1-adenosine receptors. This is in agreement with the data on transgenic left atria, where the positive inotropic effect of R-PIA or adenosine could also be blocked by DPCPX [13]. On the other hand, the increase of contractility may theoretically be explained by stimulation of A2-adenosine receptors, which are positively coupled to inotropy in some species [25]. However, using the selective A2-AR-agonist CGS21680C, we found no evidence for any positive effect of A2-AR stimulation on cell shortening, Ca2+ currents or Ca2+ transients, either in wild-type or in transgenic cardiomyocytes. Thus, stimulation of A2-adenosine receptors seems not to contribute to the paradoxical effects of R-PIA in transgenic cardiomyocytes.

The decrease in contractility in the presence of isoproterenol in wild-type preparations can be easily explained by an inhibition of cAMP generation and/or activation of protein phosphatases (for review, see Ref. [31]). Both mechanisms could account for the attenuated current through L-type Ca2+ channels [30,32]. In contrast, in transgenic mice the Ca2+ transients were not reduced but elevated (Fig. 5).

What mechanism(s) could hence underlie the positive inotropic effect of R-PIA in the presence of isoproterenol in transgenic ventricular cardiomyocytes? A possible explanation would be as follows: in transgenic cardiomyocytes, R-PIA further augmented the current through L-type Ca2+ channels stimulated by isoproterenol. This triggers Ca2+ releases from the SR and thus tends to further elevate cytosolic Ca2+. This increase was antagonized by the effect on PLB phosphorylation. R-PIA diminished isoproterenol-stimulated phosphorylation of PLB. Therefore, SERCA function is impaired and within a given time less Ca2+ is accumulated in the sarcoplasmic reticulum (SR). Subsequently, less Ca2+ is ready to be released in systole. This latter effect would cause less force development. However, there is a net increase in the level of cytosolic Ca2+ by R-PIA in the presence of isoproterenol despite the opposing effect of impaired SR-function in transgenic ventricular cardiomyocytes. Thus, additional mechanism(s) might be active in transgenic cardiomyocytes by analogy with other model systems. High concentrations of carbachol (an M-cholinoceptor agonist) can increase force of contraction in the ventricle, most probably by activation of PLC with subsequent IP3 increase [33]. A1-adenosine receptor stimulation increased IP3 content in the heart and thus might be involved in the increase of intracellular Ca2+ concentration in transgenic cardiomyocytes [34]. This question remains to be elucidated in subsequent studies.

4.2. Chronotropic and dromotropic considerations
In all mammals, A1-AR stimulation leads to a direct as well as an indirect negative chronotropic effect. This suggests that A1-adenosine receptors use different inotropic and chronotropic signalling pathways. The mechanism of chronotropic (and dromotropic) effect of A1-AR stimulation involves opening of potassium channels with subsequent hyperpolarization of nodal cells [26,27]. Despite of the paradoxical increase in force of contraction, the negative chronotropic effect of A1-AR stimulation was maintained in transgenic spontaneously beating right atrium [13]. Hypothetically, endogenous levels of adenosine were sufficient to inhibit the function of the SA node in several methodologically different preparations from transgenic animals. Bradycardia was present in isolated right atrium [13], isolated perfused hearts and conscious or anesthetized intact mice ([11]; present study). The endogenous (interstitial) levels of adenosine have not been studied in transgenic mice, but they need not to be elevated to explain the chronotropic effects we observed. Likewise, the negative dromotropic effect of endogenous adenosine [22,29] could explain the results from surface ECG (Table 2). Prolonged PQ and QRS intervals imply lower conduction rates from the SA to the AV node and slower depolarization in the ventricle and are herewith consistent with the negative dromotropic effect of adenosine.

4.3. Functional compensation of bradycardia in intact animals
We suggest a functional compensation of the bradycardia, because systolic blood pressure remained unaltered. The compensation might be a decrease in peripheral resistance (at least the coronary resistance is unaltered in isolated hearts from transgenic mice) [10,28] or increased stroke volume. The latter explanation is the most probable as contractility can be stimulated by adenosine in the cardiomyocytes from transgenic mice by A1-adenosine receptor stimulation (Fig. 3). In addition, we hypothesize that endogenous adenosine might accentuate inotropic effects of endogenous catecholamines. Therefore, the anti-adrenergic effect of adenosine is masked in transgenic hearts and it is even converted into a positive inotropic effect. Hence, cardiac contractility is probably increased in the living animal.

In conclusion, overexpression of A1-adenosine receptors in the heart reveals an increase in cellular contractility of A1-adenosine receptor stimulation in the presence of β-adrenoceptor activation. The mechanism of this effect involves an increased current through sarcolemmal L-type Ca2+ channels and increased Ca2+ transients.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the Deutsche Forschungsgemeinschaft (Ne 393/24-1) and NIH RO1 HL59419. Dr. Matherne was a recipient of an AHA Established Investigator Grant.


   Notes
 
Time for primary review 31 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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