Published online before print October 12, 2006, doi: 10.1161/01.RES.0000250046.69918.d5
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© 2006 American Heart Association, Inc.
Cellular Biology |
Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching ß1-Adrenergic but Locally Confined ß2-Adrenergic Receptor–Mediated Signaling
From the Institute of Pharmacology and Toxicology (V.O.N., M.B., E.S., M.J.L.), University of Wuerzburg; and Rudolf-Virchow-Center (S.E.), Deutsche Forschungsgemeinschaft–Research Center for Experimental Biomedicine, University of Wuerzburg, Germany.
Correspondence to Stefan Engelhardt, MD PhD, Rudolf-Virchow-Center/DFG-Research Center for Experimental Biomedicine and Martin J. Lohse, MD, Institute of Pharmacology, University of Wuerzburg, Versbacher Strasse 9, Wuerzburg 97078, Germany. E-mail stefan.engelhardt{at}virchow.uni-wuerzburg.de and lohseatoxi.uni-wuerzburg.de
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Abstract |
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ß1- and ß2-adrenergic receptors (ßARs) are known to differentially regulate cardiomyocyte contraction and growth. We tested the hypothesis that these differences are attributable to spatial compartmentation of the second messenger cAMP. Using a fluorescent resonance energy transfer (FRET)-based approach, we directly monitored the spatial and temporal distribution of cAMP in adult cardiomyocytes. We developed a new cAMP-FRET sensor (termed HCN2-camps) based on a single cAMP binding domain of the hyperpolarization activated cyclic nucleotide-gated potassium channel 2 (HCN2). Its cytosolic distribution, high dynamic range, and sensitivity make HCN2-camps particularly well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and performed single-cell FRET imaging on freshly isolated cardiomyocytes. Whole-cell superfusion with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE) inhibitors revealed stringent control of cAMP through PDE4>PDE2>PDE3. The ß1AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas ß2AR-mediated cAMP was under control of multiple PDE isoforms. ß1AR subtype–specific stimulation yielded
2-fold greater cAMP responses compared with selective ß2-subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (
FRET, 17.3±1.3% [ß1AR] versus 8.8±0.4% [ß2AR]). Treatment with pertussis toxin to inactivate Gi did not affect cAMP production. Localized ß1AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local ß2AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, ß1ARs induce far-reaching cAMP signals, whereas ß2AR-induced cAMP remains locally confined.
Key Words: cAMP • FRET • cardiomyocyte • ß-adrenergic receptor
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Introduction |
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Stimulation of cardiomyocyte ß-adrenergic receptors (ßARs) by the endogenous agonists noradrenaline and adrenaline represents the strongest mechanism to increase cardiac chronotropy and inotropy.1 The mammalian heart contains 3 ßAR subtypes: the ß1-, the ß2-, and the ß3AR. The ß1- and ß2AR subtypes dominate the cardiac response to adrenergic stimulation. Both are expressed in cardiomyocytes, couple primarily to Gs, and mediate cAMP formation, whereas coupling of the ß2AR to Gi has been described in several animal species and in failing human cardiomyocytes.2–4 The second messenger cAMP then leads to activation of protein kinase A (PKA), which phosphorylates key regulators of the cardiac excitation/contraction machinery, including the L-type Ca2+ channel, phospholamban, the ryanodine receptor, and troponin T and I. However, selective stimulation of these 2 receptor subtypes elicits different physiological responses. ß1AR stimulation, but not ß2AR stimulation, seems to induce cardiomyocyte hypertrophy.5 Transgenic mice with cardiomyocyte-specific overexpression of the ß1AR develop progressive cardiac hypertrophy and heart failure, whereas ß2AR transgenic mice do not show such abnormalities.6,7 Isolated cardiomyocytes undergo apoptosis on ß1-selective stimulation, and ß2 stimulation may protect against this.8,9 Furthermore, differences in the PKA-mediated phosphorylation pattern are observed after subtype-specific stimulation, and the ßAR subtypes behave differently regarding the inhibition of muscarinic receptor signaling and the activation of CNG channels.10–12 These findings cannot be sufficiently explained by differential coupling of ß1ARs and ß2ARs to Gs and Gi proteins. In addition, work by Xiao, Lakatta, and colleagues indicated differential compartmentation of ß1AR- and ß2AR-mediated cAMP signaling.13–16 Differences between ß1- and ß2AR signaling have been interpreted mainly through compartmentation of signaling events, such as the formation of signalosomes17,18 and the localized control of cAMP degradation through phosphodiesterases (PDEs).19,20 However, visualization of localized cAMP in cardiomyocytes has been difficult, because of the lack of appropriate techniques.
Recently, the advent of fluorescence resonance energy transfer (FRET) has allowed the development of protein-based sensors to observe intracellular signaling events in real time. Several sensors have been described that exhibit FRET changes on exposure to cAMP.21 A sensor based on the dissociation of 2 PKA subunits was first described by Adams et al (using rhodamine and fluorescein as fluorophores)22 and later modified Zaccolo and colleagues to be genetically encoded by using green fluorescence protein (GFP) variants instead of rhodamine and fluorescein.23,24 This sensor has led to major insights into the biology of cAMP.25–27 Recently, it has been introduced into adult cardiomyocytes via adenoviruses.25,27 However, PKA-based sensors have several important limitations for the study of adult cardiac myocytes. The catalytic domain of PKA is enzymatically active and displays significant toxicity when expressed in various cell types. Importantly, PKA-based sensors are strictly localized in cardiomyocytes through binding to A-kinase anchoring proteins (AKAPs),17,19 thus making it difficult to decide whether localized FRET signals are attributable to localization of cAMP or of the localized sensor. Finally, this strategy necessitates the cotransfection of 2 different proteins at equal concentrations in a single cell. We have recently described another sensor, based on the single cAMP binding domain of Epac.28 This sensor is monomeric and has the advantage of homogeneous distribution throughout the cell. However, both PKA- and Epac-based sensors have the inherent disadvantage that their sensitivity is relatively high (affinity for cAMP, 1 µmol/L).28,29 Therefore, the determination of cAMP with these constructs is limited to a certain range of lower cAMP concentrations, which seems to be exceeded in adult cardiomyocytes.
Therefore, we have developed a novel sensor with optimized sensitivity based on a single cAMP binding domain of the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2). We have expressed this sensor in a cardiomyocyte-specific manner in the hearts of transgenic mice and studied spatial and temporal cAMP dynamics after ß1- and ß2AR-subtype stimulation in freshly isolated adult cardiomyocytes.
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Materials and Methods |
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Molecular Biology, Cell Culture, and Western Blot Analysis
To generate the novel cAMP sensor HCN2-camps, the DNA encoding for a single cAMP binding domain of the murine HCN2 channel (amino acid range A467 to K638) was amplified from a heart cDNA library by PCR and subcloned into a pcDNA3-based vector between enhanced yellow (EYFP) and enhanced cyan (ECFP) fluorescent protein. Western blot analysis was performed as described previously.30
Transgenic Mice and Cardiomyocyte Isolation
For the generation of transgenic mice, the HCN2-camps sequence was cloned into pB–
-MHC vector between KpnI and EcoRV restriction sites. The isolation of adult mouse cardiomyocytes was performed essentially as described previously.30 After calcium adaptation, the cells were seeded onto laminin-coated coverslips.
Measurements of FRET and Fluorescence Recovery After Photobleaching
A detailed description of the optical methods can be found in the online data supplement, available at http://circres.ahajournals.org.
Statistical Analysis
Average data are presented as mean±SEM. Statistical analysis was performed using the Prism software package (GraphPad, San Diego, Calif). Statistical significance was evaluated using Student’s t test. P<0.05 was considered statistically different.
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Results |
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Functional Characterization of the HCN2-Based cAMP Sensor
Basal cAMP levels in cardiomyocytes have been reported to be higher compared with some other cell types.31,32 To create a fluorescent sensor that would be optimally suited to report agonist-induced cAMP changes in cardiomyocytes, we sought to extend the dynamic range of existing single-domain FRET sensors toward higher cAMP concentrations. We took a single cytoplasmatic cAMP binding domain of HCN2 channel endogenously expressed in the heart as a backbone for a novel cAMP sensor, which we termed HCN2-camps. Based on the crystal structure of HCN2,33 we fused its cAMP binding domain between YFP and CFP to monitor cAMP-dependent conformational changes by FRET between the 2 fluorescent proteins (Figure 1a).
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HCN2-camps isolated from transiently transfected HEK293a cells demonstrated micromolar affinities for cAMP (Figure 1b) (EC50, 5.9±0.3 µmol/L; n=4), whereas cGMP activated the sensor only partially at high micromolar concentrations (maximum, 31.5±2.3% of cAMP signal at 600 µmol/L). Next, we expressed HCN2-camps in HEK293a cells to monitor agonist-dependent changes in FRET reflecting cAMP synthesis and degradation. Addition of the ßAR agonist isoproterenol (Iso) resulted (via stimulation of cAMP production by endogenous ß2ARs) in a rapid decrease in FRET (reflecting a rise in intracellular cAMP), which was reversible on withdrawal of the ligand (Figure 1c). In contrast to some previously developed FRET-based cAMP sensors (PKA-based probes,23,24 Epac-camps28), HCN2-camps retained a high sensitivity but did not show any plateau at high physiological cAMP concentrations, reflecting saturation of the sensor. These properties of HCN2-camps suggest a high dynamic range of this sensor and its suitability to monitor both low and high cAMP concentrations.
Expression of HCN2-Camps in Mouse Heart
To be able to work with freshly isolated cells, we generated transgenic mice expressing the sensor in a cardiomyocyte-specific manner under control of the -myosin heavy chain (-MHC) promoter (Figure 2a).
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Pronuclear injection of the construct into oocytes yielded 2 transgenic founder animals, as revealed by PCR analysis (Figure 2a, bottom), which gave rise to 2 independent mouse lines, termed TG5 and TG10. The animals appeared healthy and had a normal life expectancy. The hearts of mice expressing HCN2-camps did not show any visible morphological alterations (not shown). Both transgenic lines TG5 and TG10 demonstrated approximately equal expression of the sensor protein in the heart confirmed by Western blot experiments on heart lysates (Figure 2b).
Next, we isolated adult ventricular myocytes from transgenic hearts and analyzed fluorescence of single cells for the presence of CFP and YFP emissions. Wild-type cardiomyocytes showed only weak autofluorescence at high exposure times, whereas no fluorescence signal was detectable at the 10-ms exposure time used for the detection of FRET. Virtual, all cells isolated from transgenic mice of both lines displayed a strong fluorescence in both CFP and YFP channels (50 times above autofluorescence), indicating efficient expression and a excellent signal-to-background ratio of HCN2-camps expressed in cardiomyocytes. Ratio images of the cells showed high basal FRET levels and uniform distribution of the signal throughout the cells (Figure 2c).
To assess a potential influence of HCN2-camps diffusion on the determination of cAMP diffusion, we tested how fast the HCN2-camps protein diffuses in cardiomyocytes using a fluorescence recovery after photobleaching (FRAP) approach (Figure 2d). Exponential analysis of fluorescence recovery yielded a diffusion coefficient of 7 µm2/sec, which corresponds well to free diffusion of a small protein.
Dynamic Changes of cAMP in Adult Cardiomyocytes After Adrenergic Stimulation
To test whether HCN2-camps reports agonist-induced cAMP changes in cardiomyocytes, we first superfused them with the unselective ßAR agonist Iso, which produced a moderate cAMP elevation (Figure 3a). Under stimulation with Iso (Figure 3a) or the natural agonist norepinephrine (NE), no obvious striated pattern of fluorescence ratio change was observed. FRET signals recorded by the HCN2-camps sensor appeared uniformly distributed throughout the cytosol of adult cardiomyocytes. Subsequent treatment of cells with the PDE4 inhibitor rolipram resulted in a further significant elevation of cAMP levels, reversible on washout of the inhibitor and Iso (Figure 3a).
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The concentration-response relations measured with HCN2-camps for Iso and NE are presented in Figure 3b and show EC50 values within the expected concentration range of these ligands.
Next, we analyzed the contributions of different PDE isoforms to cAMP hydrolysis in adult cardiomyocytes (Figure 3c through 3f). Addition of selective inhibitors of all major PDE isoforms expressed in the heart (PDE2–erythro-9-(2-hydroxy-3-nonyl) adenine [PDE2-EHNA] 10 µmol/L, PDE3-milrinone 1 µmol/L, and PDE4-rolipram 100 nmol/L) or of the unselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) (300 µmol/L) induced further elevation of intracellular cAMP when applied together with Iso, although to different extents. The highest cAMP hydrolyzing activity after adrenergic stimulation was attributable to PDE4, whereas PDE2 and PDE3 were significantly less active (Figure 3f). In contrast, PDE inhibition did not produce any significant FRET change of HCN2-camps in unstimulated cells (Figure 3f). Glucagon has been documented to elevate cardiomyocyte cAMP in the absence of a positive inotropic effect. Compared with the robust adrenergic effect, glucagon (1 µmol/L) led to a more modest elevation of cAMP (1.48±0.14%; n=5).
Differential cAMP Signaling Profiles Under ß1- and ß2AR Stimulation
To investigate how different ßAR subtypes regulated cAMP dynamics in adult cardiomyocytes, we used selective stimuli to activate either ß1ARs (combination of Iso with the ß2AR antagonist ICI118551) or ß2ARs (Iso with ß1AR antagonist CGP20712A). First, we tested whether these antagonists might affect cAMP levels per se. ICI118551 (5 nmol/L) and CGP20712A (100 nmol/L) did not show any significant effect on HCN2-camps FRET ratio (change in FRET 0.17±0.09% and 0.01±0.15%, respectively; n=4).
ß1AR stimulation led to a 4.2±0.4% change in FRET (Figure 4a and 4c) comparable to stimulation with Iso alone (4.3±0.3%; Figure 3f). Combination of ß1AR selective stimulation with rolipram led to a 4-fold increase of the FRET signal; the same effect was achieved by the nonselective PDE inhibitor IBMX, indicating that PDE4 is the major isoform to restrict cAMP accumulation evoked by ß1AR stimulation (Figure 4a and 4c).
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In contrast, selective stimulation of ß2ARs only led to a 2.3±0.3% change in FRET (Figure 4b and 4c), which is almost 2-fold less than on ß1AR stimulation. We next tested how different PDE isoforms affect cAMP dynamics after ß2AR stimulation. Either rolipram or milrinone applied together with Iso/CGP equally increased intracellular cAMP (to 5% change in FRET), thereby achieving ß1AR-induced levels in the absence of PDE inhibitors (Figure 4b). Treatment of cardiomyocytes with a combination of both rolipram and milrinone or the nonselective PDE inhibitor IBMX further increased the cAMP signal on ß2AR stimulation. However, the changes in FRET never reached more than 50% of ß1AR-induced cAMP levels in the presence of PDE inhibitor treatment (Figure 4c).
Finally, we tested the possibility that cAMP production by ß2ARs might be restricted by its coupling to Gi inhibitory G proteins. Incubation of cardiomyocytes with pertussis toxin (PTX) for 20 hours led to complete inhibition of carbachol-mediated decrease in intracellular cAMP but failed to increase cAMP production on ß2AR stimulation (2.5±0.1% change in FRET; Figure 4c). The data on the effects of PDE inhibitors and PTX suggest that additional PDE- and Gi-independent mechanisms might restrict cAMP diffusion after ß2AR stimulation.
To test how cAMP responses are spatially organized, we used localized stimulation of cardiomyocytes with a micropipette (Figure 5a) and analyzed intracellular cAMP propagation after receptor stimulation. Localized stimulation of ß1ARs evoked cAMP diffusion gradients propagating throughout large parts of a adult cardiomyocyte (Figure 5b and 5e). From these experiments, we calculated the speed of cAMP propagation. To do so, we fitted the ratio traces with a monoexponential curve for adjacent regions of a cell and determined the time points where this curve crossed the baseline (Figure 5b). The speed of cAMP diffusion was thereby determined to amount to 15.6±2.1 µm/sec (n=8), from which the diffusion coefficient of cAMP in adult cardiomyocytes was derived (136.3±36.1 µm2/sec). In contrast to the propagating cAMP gradients from ß1ARs, stimulation of ß2ARs by a pipette induced only a localized elevation of cAMP, which did not propagate throughout the cell (Figure 5d and 5e). Even in the presence of PDE inhibitors (rolipram plus milrinone; data not shown), no propagation of ß2AR-induced cAMP was observed. In both cases, responses to local ß1- and ß2AR stimulation did not show any striated pattern of the FRET ratio.
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We wondered whether the absence of a propagating cAMP gradient after ß2AR stimulation might be caused simply by the lower expression density of this receptor subtype compared with the ß1AR. To test this possibility, we blocked a large fraction of the ß1AR by a nonsaturating CGP20712A concentration, thus achieving a ß1AR-mediated cAMP signal that closely mimics the amount of cAMP produced after selective stimulation of the ß2AR subtype (FRET, 2.3±0.3%). Under these conditions, the cAMP gradient caused by selective ß1AR stimulation still propagated throughout the cardiomyocyte, even though the amplitude of the signal was reduced (Figure 5c and 5e).
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Discussion |
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These data represent the first direct visualization of the differences between ßAR-subtype signaling in cardiomyocytes. We describe a novel FRET-based sensor for the detection of cAMP that is homogenously distributed throughout the cytosol. Determination of cAMP in adult cardiomyocytes after ßAR subtype–specific stimulation yielded crucial differences of cAMP signaling between the 2 receptor subtypes. ß1AR-mediated cytoplasmic cAMP is mainly controlled by PDE4, whereas cAMP produced after ß2AR stimulation is under the control of multiple PDE isoforms. Under native conditions, ß1AR-mediated cAMP diffuses over more than 20 µm, whereas the ß2AR signal remains restricted to local domains.
The uniform distribution of HCN2-camps inside the cell makes it ideally suited to address the issue of localized versus nonlocalized cAMP compartments. The local recording of plasma membrane bound ion channels12,34 and PKA-based sensors targeted via AKAPs to discrete intracellular locations24 gives information primarily about the specific subcellular regions, where the respective sensors are located. This yielded important insights about the dynamics of subsarcolemmal cAMP pools12,34 and cAMP signaling in the vicinity of AKAP-containing protein complexes.24,25,27 In contrast, the homogenous distribution of HCN2-camps allows the recording of cAMP signals throughout the whole cell.
Our data recorded with HCN2-camps demonstrate that cAMP in cardiomyocytes diffuses over longer distances than previously thought. Although in neonatal cardiomyocytes, a range of 2 µm has been described, which suggests that the range of a local cAMP signal is restricted entirely to the individual sarcomere,24 we routinely observed ranges up to 30 µm in adult cardiomyocytes after adrenergic stimulation. We cannot rule out, at this point, that this is attributable to the use of adult versus neonatal cells; however, the highly ordered structure of freshly isolated adult cardiomyocytes renders this possibility unlikely. Another explanation for these discrepancies could be the use of localized versus nonlocalized sensors. Homogenously distributed HCN2-camps in this case might just diffuse over long distances in cells, showing a propagating signal. To assess the diffusion of the sensor in cardiomyocytes, we performed FRAP experiments (Figure 2d), which yielded a diffusion coefficient of 7 µm2/sec for HCN2-camps. Compared with the speed of cAMP propagation measured in Figure 5b and calculated at 136 µm2/sec, this value is at least 19 times slower, excluding the possibility that the propagating gradients measured with HCN2-camps after localized cardiomyocyte stimulation reflect the diffusion of the sensor.
Using electrophysiological recordings of submembrane cAMP by adenovirally transfected CNG channels, ß1- and ß2ARs were recently shown to yield different cAMP-mediated effects (absence of any response on ß2AR stimulation and a 3-fold increase in current on ß1AR stimulation), a difference that was abolished in presence of IBMX (equal responses for ß1- and ß2ARs) but detectable in cell lysates by radioimmunoassay.12 In contrast, our approach allows to extend the determination of cAMP into the cytosol, where ß1AR and ß2AR-mediated cAMP signals differ markedly. In the absence of PDE inhibition, we observe a twofold greater cAMP response after ß1AR stimulation compared with ß2AR stimulation. Relieving the PDE-mediated control of cAMP, the ß2AR cAMP signal is still 2-fold smaller, suggesting that the diffusion of cAMP from the submembrane compartment into the cytosol is restricted by additional mechanisms. These might involve the differential localization of the individual receptor subtypes with respect to caveolae and to cardiomyocyte t-tubular structures.18,35 Additional mechanisms that could potentially account for the weaker ß2AR-mediated response in the whole-cell configuration include differences in expression levels and G-protein coupling of the individual ßAR subtypes.3 We tested whether Gi coupling of the ß2AR affects cAMP production through preincubation of our cells with PTX. However, inactivation of Gi did not increase the ß2AR-mediated cAMP formation (Figure 4c).
Another major difference setting our model apart from previous reports25,27 is the use of freshly isolated adult cardiomyocytes. In our hands, adenoviral expression and proper folding of sensors usually necessitates prolonged periods of culture. This regularly involves remodeling of the intricate t-tubular network of adult cardiomyocytes.36 Because the complex intracellular architecture of cardiomyocytes is likely to play an important role in compartmentation of cAMP signaling, our experimental strategy aimed at preserving the native structure of the adult cell. To be able to work with freshly isolated cells, we have introduced HCN2-camps into cardiomyocytes by creating transgenic mice expressing the sensor in a cardiomyocyte specific manner.
Our data underline the importance of PDE isoforms for the control of cardiomyocyte cAMP and extend previous findings.12,24,29,34 The ß1AR and the ß2AR differ markedly as to the PDE isoforms involved in the control of their cAMP signals. Whereas in the submembrane compartment, both PDE3 and PDE4 in concert are necessary to control ß2AR-mediated cAMP,12 our data support both an independent and additive role for PDE3 and PDE4 (Figure 4c). Measurements of cAMP in neonatal cardiomyocytes using a PKA sensor localized to AKAP-induced signaling complexes revealed an increase of cAMP after addition of PDE inhibitors to unstimulated cells,24,29,37 whereas HCN-camps localized to the cytosol did not report such effects compatible with CNG-based measurements in adult rat cardiomyocytes.12 A reason for this discrepancy could be the use of neonatal versus adult cells. However, the data of different studies suggest that the submembrane and the cytosolic cAMP compartments exert different mechanisms of cAMP control. On the other hand, our data obtained with HCN-camps differ from the CNG-based method, which does not allow to detect a significant cAMP signal after selective ß2AR stimulation unless PDE activity is inhibited.12 This suggests that the ß2AR signal detected by HCN2-camps does not originate from the compartment the CNG method allows to assess. Although we do not know the exact localization of the ß2AR and that of CNG channels on the cardiomyocyte surface at this point, it might be possible that ß2ARs and CNG channels reside on different compartments.
Local stimulation of ß1ARs resulted in a cAMP signal, which propagated inside adult cardiomyocytes over a distance spanning multiple sarcomeres. In contrast, the ß2AR-induced signal did not propagate over long distances throughout the cell. Several lines of evidence indicate that these differences cannot be explained by a weaker expression of ß2ARs, which constitute approximately 20% to 30% of the whole ßAR population in adult mouse cardiomyocytes. We have blocked the majority of ß1ARs during ß1AR-selective stimulation (Figure 5c). We thereby reduced the ß1AR-induced FRET signal to 2%, thus closely mimicking the cAMP levels achieved through ß2AR stimulation. Also under these conditions, ß1AR-induced, but not ß2AR-induced, cAMP propagated throughout the cell (Figure 5c and 5d). In addition, ß2AR-mediated cAMP did not propagate under inhibition of PDE3 and -4 (data not shown), whereas the amounts of cAMP produced under these conditions in the whole-cell configuration exceeded those of ß1AR stimulation (Figure 4c).
Our experiments suggest that also PDE-independent mechanisms are involved in cAMP compartmentation. These could involve physical restriction of cAMP diffusion, for example, elicited through differential localization of the individual ßAR subtypes on the surface of adult cardiomyocytes.
Taken together, ß1- and ß2ARs elicit distinct cAMP responses, which are under control by various PDE subtypes. Local ß1AR-mediated cAMP signals propagate over a distance involving multiple sarcomeres in adult cardiomyocytes. In contrast, the ß2AR-evoked cAMP signal remains strictly confined by PDE- and Gi-independent mechanisms.
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Acknowledgments |
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The expert technical assistance of Silke Oberdorf-Maass and Julia Schittl is gratefully acknowledged. We thank Francesca Rochais for critical reading of the manuscript.
Sources of Funding
This work was funded by the Rudolf-Virchow-Center/FG –Research Center for Experimental Biomedicine supported by ProCorde, Sanofi-Aventis, and the Bavarian Futher support came from Ministry of Economics, the Leducq Foundation (M.J.L. and S.E.), and the Bundesministerium für Bildung und Forschung (German Heart Failure Network to S.E.). M.J.L. and M.B. were supported by the Deutsche Forschungsgemeinschaft (Ernst-Jung-Award and grant SFB688).
Disclosures
None.
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Footnotes |
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Original received July 28, 2006; revision received October 4, 2006; accepted October 4, 2006.
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References |
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