- Email: [email protected]
Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle
Life Sciences 88 (2011) 892–897
Contents lists available at ScienceDirect
Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle Joaquín Pérez-Schindler a,⁎, Andrew Philp b,1, Keith Baar b,1, Jesús Hernández-Cascales a a b
Department of Pharmacology, Faculty of Medicine, University of Murcia, Murcia, Spain Division of Molecular Physiology, University of Dundee, Dundee, UK
a r t i c l e
i n f o
Article history: Received 7 September 2010 Accepted 25 March 2011 Keywords: β2-adrenergic receptor Cardiac contractility Cardiac metabolism AMPK PI3K
a b s t r a c t Aims: Cardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (βAR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β2-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β2-AR activation by tulobuterol. Main methods: Strips of rat right ventricle were electrically stimulated (1 Hz) in standard Tyrode solution (95% O2, 5% CO2) in the presence of the β1-antagonist CGP-20712A (1 μM). A cumulative dose–response curve for tulobuterol (0.1–10 μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30 μM), or 10 min incubation (1 μM) with the β2-agonist tulobuterol was performed. Key findings: β2-AR stimulation induced a positive inotropic effect (maximal effect = 33 ± 3.3%) and a decrease in the time required for half relaxation (from 45 ± 0.6 to 31 ± 1.8 ms, − 30%, p b 0.001) after the inhibition of PDEs. After 10 min of β2-AR stimulation, p-AMPKαT172 (54%), p-PKBT308 (38%), p-AS160T642 (46%) and p-CREBS133 (63%) increased, without any change in p-PKAT197. Significance: These results suggest that the regulation of ventricular contractility is not the primary function of the β2-AR. Rather, β2-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle. © 2011 Elsevier Inc. All rights reserved.
Introduction The contractile and metabolic proprieties of cardiac muscle are critical factors in the regulation of heart performance and the susceptibility to cardiac dysfunction (Hill and Olson, 2008). Heart function is modulated by the sympathetic nervous system through βadrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. In the heart, the β1 and β2-AR are the main receptors expressed (Salazar et al., 2007) and are each thought to activate alternate signaling pathways and regulate diverse cellular responses (Xiao et al., 2004). Stimulation of the β2-AR exerts a cardioprotective effect, whereas β1-AR stimulation has a negative effect on cardiac function (Bernstein et al., 2005; Xiao et al., 2004; Zhu et al., 2001). The β1-AR is the most abundant subtype in the heart and the main regulator of cardiac contractility (Salazar et al., 2007; Yoo et al., 2009). β1-AR modulates cardiac function via the stimulatory G protein (Gαs)/
⁎ Corresponding author at: Focal Area Growth and Development, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland. Tel.: + 41 61 267 2237; fax: + 41 61 267 2221. E-mail address: [email protected] (J. Pérez-Schindler). 1 Current address: Neurobiology, Physiology and Behavior, University of California, Davis, CA, United States. 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.03.020
adenylyl cyclase (AC)/cyclic AMP (cAMP)/cAMP dependent protein kinase (PKA) signal pathway, leading to the phosphorylation of proteins involved in regulating excitation-contraction coupling in cardiac muscle (Salazar et al., 2007). β2-AR is coupled not only to Gαs but to an inhibitory G protein (Gαi), that can block AC and prevent PKA activation (Xiao et al., 1995). This dual coupling to Gαs and Gαi is consistent with the lower inotropic response reported in rat heart following administration of β2 compared to β1-AR agonists (McConville et al., 2005). Therefore, the regulation of cardiac muscle contractility does not seem to be the primary function of β2-AR in rodent heart. In addition to inhibiting Gαs/AC/PKA, Gαi has other functions within the heart. Together with the Gβγ heterodimer, Gαi can activate phosphatidylinositol 3-kinase (PI3K) (Jo et al., 2002). In fact, some β2agonists induce hypertrophy of cardiac and skeletal muscle (Lynch and Ryall, 2008; Pönicke et al., 2003) and prevent apoptosis in a Gαi/ PI3K dependent manner (Communal et al., 1999; Zhu et al., 2001). In association with the increase in cardiac muscle mass, β2agonists also affect muscle metabolism. Soppa et al. (2005) demonstrated that chronic treatment with the β2-agonist clenbuterol increased carbohydrate (CHO) contribution to the tricarboxylic acid (TCA) cycle in rat ventricular cardiomyocytes, whereas the stimulation of β2-AR in skeletal muscle increases fatty acid (FA) oxidation while glucose uptake is diverted towards glycogen synthesis (Nevzorova et al., 2006; Ngala et al., 2008; Yamamoto et al., 2007).
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897
Collectively, these studies provide evidence that the β2-AR could play an important role in the regulation of cardiac metabolism, however, to date, the cellular signaling pathways involved in these responses are not well characterized. Chronic obstructive pulmonary disease (COPD) is associated with the development of pulmonary hypertension, which impairs right ventricle function by inducing pathological hypertrophy (MacNee, 2010). Therefore, the assessment of the impact of β2-agonists on right ventricle function has a particular interest for the treatment of COPD. Tulobuterol is a β2-agonists used for long-term treatment of COPD in humans (Patel, 1985). Besides early pharmacological studies (GonzalezSicilia et al., 1988; Laorden et al., 1985; Ruff et al., 1988), the effects of tulobuterol on cardiac function have not been examined. Therefore, the aim of the present study was to determinate the effect of selective β2-AR stimulation with tulobuterol on contractility and intracellular signaling in rat right ventricle preparations ex vivo.
893
single dose of 1 μM tulobuterol for 10 min. On completion, cardiac strips were frozen in liquid nitrogen and stored at − 80 °C until analysis. Protein isolation Samples were pulverized on dry ice, homogenized with a polytron in 200 μL of ice cold sucrose lysis buffer (50 mM Tris pH 7.5, 250 mM Sucrose, 1 mM EDTA, 1 mM EGTA, 1% Triton X 100, 50 mM NaF, 1 mM Na3VO4, 50 mM Na4P2O7, 0.1% DTT) and shaken for 30 min at 4 °C. Samples were then centrifuged at 14 000 rpm for 10 min at 4 °C and the protein concentration of the supernatant determined by the DC protein assay (Bio-Rad, Hercules CA). Equal aliquots of protein were boiled for 5 min in Laemmli sample buffer (250 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 5% β-mercaptoetanol). Western blot
Materials and methods The study was performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and approved by the Ethical Committee of the University of Murcia. Animals and right ventricle isolation Male adult Sprague–Dawley rats (250–300 g) were killed by a blow to the head followed by cervical dislocation. The heart was rapidly removed and placed in Tyrode solution (136.9 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1.5 mM MgCl2, 0.4 mM NaH2PO4, 11.9 mM NaHCO3, 5 mM D-glucose and 5 mM pyruvate) maintained at pH 7.4 and gassed with 95% O2 and 5% CO2. The external wall of the right ventricle was extracted and cut into strips ~1 mm wide, ~10 mm long and ~ 0.5 mm thick. Subsequently, the strips were mounted longitudinally between two platinum electrodes, placed in an organ bath (30 mL) with Tyrode solution (37 °C, pH 7.4, 95% O2 and 5% CO2) and electrically stimulated (Grass SD-9) against a preload of ~ 1 g at 1 Hz, 5 ms and supramaximal voltage (~ 25% above response threshold). Contraction amplitude and relaxation was measured with a force transducer (Grass FT-03) and amplified (Stemtech Inc., Houston, Texas) for future quantification. The strips were allowed to equilibrate with the experimental conditions for ~40 min before drug treatment. Cumulative dose–response curve After equilibration, a cumulative dose–response curve with the β2agonist tulobuterol (0.1–10 μM; Lab. Ferrer, Co. Spain) in the presence of the β1-antagonist CGP-20712A (1 μM; Sigma) was performed. To determinate the selective β2-AR activation, the β2-antagonist ICI118,551 (50 nM; Sigma) was used. The role of the cyclic nucleotide phosphodiesterase (PDE) was determined using the non selective PDE inhibitor IBMX (30 μM; Sigma). The β-antagonists and IBMX were added to the bath 15 min before tulobuterol addition. Drugs were added to the organ bath (30 mL) in a volume no higher than 0.1 mL. The experiments were finalized with the addition of 9 mM CaCl2 to determinate the response capacity and maximal contractile amplitude of the preparations. Changes in contractile force induced by tulobuterol are expressed as a percentage of the control contraction amplitude (in the presence of the corresponding β-antagonist and IBMX, if applicable). The relaxation capacity of the strips was studied by the determination of the time required for half relaxation (t1/2). Incubation for protein phosphorylation measurement To determine the metabolic signaling regulated in response to the selective β2-AR activation, the strips were incubated after the equilibration with 1 μM CGP-20712A alone or in combination with a
Samples were separated on an SDS-polyacrylamide gel (8.25%) for 50 min and then transferred to a Protran nitrocellulose membrane (Whatman, Dassel, Germany) for 60 min. Membranes were blocked for 1 h in 3% milk in Tris-buffered saline +0.1% tween-20 (TBST) before overnight incubation at 4 °C with appropriate primary antibody in TBST (1:1000 dilution). Proteins were detected with a primary antibody to p-PKBT308 (Cell Signaling; 4056), p-AMPKαT172 (Cell Signaling; 2535), p-CREBS133 (Rockland; 600-401-270), p-PKAT197 (Cell Signaling; 4781), the primary antibodies to p-AS160T642 were kindly provided by Professor Grahame Hardie (Division of Molecular Physiology, University of Dundee). Since the tulobuterol treatments lasted only 10 min and therefore the total amount of the signaling proteins was unlikely to change, eEF2 (Cell Signaling; 2332) was used as a loading control for all western blots. Following incubation, membranes were washed 3 times with TBST before incubation with an appropriate peroxidase-conjugated secondary antibody in TBST (1:10 000 dilution; Pierce, Rockford, IL). Antibody binding was detected using enhanced chemiluminescence HRP substrate detection kit (Millipore, Billerica, MA). Imaging and band quantification were carried out using a ChemiGenius Bioimaging Gel Doc System (Syngene, Cambridge, UK). Statistical analysis All values are expressed as mean ± S.E.M (n = 4–5 independent experiments). The log EC50 was calculated by the analysis of the non linear regression of the dose–response curve (Graph Pad Software, San Diego, CA). The statistical significance was determined using the Student t-test for paired and unpaired data. The significance difference was set at p b 0.05. Results Inotropic and lusitropic effect of the selective β2-AR stimulation The β-antagonists and IBMX did not alter contraction amplitude compared to basal levels (data not shown). In addition to its effects on β2-AR stimulation, tulobuterol has also been suggested to stimulate β1-AR at high concentrations (Morin et al., 2000). To ensure that the effects of tulobuterol were β2-AR specific, the β1-antagonist CGP20712A was used in combination with tulobuterol. Thus, tulobuterol in the presence of CGP-20712A did not produce a positive inotropic effect (n = 4; Fig. 1A and B). This data provides preliminary evidence that activation of the β2-AR does not increase the contractile force of the heart. However, IBMX in combination with CGP-20712A produced a dose-dependent positive inotropic effect in response to tulobuterol, with a maximal effect of 33 ± 3.3% and a log EC50: − 6.9 ± 0.6 M (n = 5; Fig. 1A and B). Moreover, under these conditions, tulobuterol not only increased the contractile force but also induced a decrease in
894
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897
by the β2-antagonist ICI-118,551 (n = 4; Fig. 1A and B), suggesting that the β2-AR is required for positive inotropy under these experimental conditions. PKA and CREB regulation by the β2-AR To induce selective β2-AR stimulation and explore the intracellular signaling mediated by this receptor, ventricular strips were incubated with the β1-antagonist CGP-20712A alone or in the presence of tulobuterol. After ten minutes of selective β2-AR stimulation with tulobuterol, there was no change (p = 0.09) in the phosphorylation of PKAT197 (n= 5; Fig. 3). In contrast to its effects on PKA, tulobuterol induced a significant increase (63%; n = 4, p b 0.01) in the phosphorylation of CREBS133 (Fig. 3). Metabolic signaling induced by the β2-AR Tulobuterol in the presence of CGP-20712A increased the phosphorylation of AMPKαT172 by 54% (n = 5, p b 0.001; Fig. 4). Additionally, 10 min of incubation with tulobuterol in the presence of
Fig. 1. Inotropic response to the β2-agonist tulobuterol. Representative experiment (A) and average values (B) of the modification of contractile force in response to tulobuterol (0.1-10 μM) in the presence of 1 μM CGP-20712A alone (n= 4, CGP20) or in combination with 30 μM IBMX (n= 5, CGP20 + IBMX) or in addition to 50 nM ICI-118,551 (n= 4, CGP20 + IBMX + ICI). Each point represents the average ±S.E.M. Data was analyzed by unpaired Student t-test. **p b 0.01, ***p b 0.001 vs. CGP20 and CGP20 + IBMX+ ICI.
the t1/2 from 45 ± 0.6 to 31 ± 1.8 ms (n = 5, − 30%, p b 0.001; Fig. 2) suggesting that PDE need to be inhibited before β2-agonists increase inotropy. In support of this, positive inotropy was completely blocked
Fig. 2. Lusitropic response to the β2-agonist tulobuterol. Representative experiment (A) and average values (B) of the decrease in the time required to the half relaxation time (t1/2) in the presence of 1 μM CGP-20712A combined with 30 μM IBMX alone (n= 5, CON) or in response to 3 μM tulobuterol (n= 5, TUL). Each bar represents the average±S.E.M. Data was analyzed by paired Student t-test. ***p b 0.001 vs. CON.
Fig. 3. Regulation of downstream proteins of the β2-AR. PKA (n = 5) and CREB (n = 4) phosphorylation in the presence of 1 μM CGP-20712A alone (CON) or in response to 1 μM tulobuterol (TUL) during 10 min incubation. Each bar represents the average ±S.E.M. Data was analyzed by paired Student t-test. **p b 0.01 vs. CON.
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897
CGP-20712A resulted in an increase in the phosphorylation of PKBT308 (n = 5, 38%, p b 0.05; Fig. 4). Along with the changes in AMPK and PKB phosphorylation, the phosphorylation of their downstream target
895
AS160T642 was significantly increased in the tulobuterol treated strips (n = 5, 46%, p b 0.01; Fig. 4). The phosphorylation of p38T180/Y182 decreased − 8.6% (data not shown) in response to the selective activation of β2-AR with tulobuterol, suggesting that the p38 MAPK pathway is not a target for Tulobuterol. Discussion The results obtained in the present study demonstrate that the β2AR directly regulates metabolic signaling, however the β2-AR does not appear to modulate contractility in rat ventricular muscle without concomitant inhibition of PDE. Selective β2-AR stimulation with tulobuterol did not enhance the contractile force of ventricular strips, but did produce an increase in the phosphorylation of AMPKαT172, PKBT308 and their downstream targets AS160T642 and the transcription factor CREBS133, suggesting a direct link between β2-AR and intracellular signaling associated with the modulation of energetic metabolism and cardiac muscle plasticity. The β2-agonist tulobuterol is a bronchodilator characterized by an EC50 of 0.15 μM, an intrinsic efficacy of 35% of isoproterenol and a halflife of 3.1 h (Kume, 2005; Patel, 1985; Terpstra and Raaijmakers, 1990). While tulobuterol weakly stimulate β1-AR in rat brain (Morin et al., 2000), in the heart it has a minimal effect on β1-AR and cardiac contractility (Gonzalez-Sicilia et al., 1988; Laorden et al., 1985; Ruff et al., 1988). The absence of a positive inotropic effect in response to selective β2-AR stimulation with tulobuterol is consistent with previous reports that show that the positive inotropic effect of β2agonists (e.g. fenoterol) can be blocked by CGP-20712A (GonzalezMuñoz et al., 2009; Siedlecka et al., 2008). This would suggest that the variability in the inotropic effect induced by different β2-agonists is, at least in part, mediated by the activation of the β1-AR. Therefore, given the deleterious effects of β1-AR stimulation on heart function, tulobuterol could be a more attractive therapeutic drug in comparison with other β2-agonists. The contractile effect of selective β2-AR stimulation was only observed when PDEs (Fig. 1) or Gαi (Gonzalez-Muñoz et al., 2009) were inhibited, resulting in positive lusitropy and a dose-dependent inotropic effect. This suggests that the contractile effects of β2-AR stimulation are dependent on cAMP production and degradation and provide evidence of a critical role for PDEs in modulating these responses. However, both Gαi and PDEs are functionally active in normal cardiomyocytes and induce a negative effect on Gαs signaling and this may explain why contractile force and PKA phosphorylation did not increase in response to selective β2-AR activation with tulobuterol. Tulobuterol in the presence of the CGP-20712A increased the phosphorylation of PKBT308, which is a key downstream protein in the PI3K signaling pathway (Latronico et al., 2004), further supporting a role for PI3K in the β2-AR signaling. In ventricular cardiomyocytes, PI3K can blunt the inotropic response to both β1 and β2-AR stimulation (Jo et al., 2002; Leblais et al., 2004). The possible mechanism by which PI3K exerts this effect on contractility could be through the activation of PDEs and the resultant decrease in cAMP (Gregg et al., 2010). Indeed, β-ARs are functionally coupled to PDEs (Rochais et al., 2006) and PDEs can be activated by β-AR (Leroy et al., 2008) and PI3K (Gregg et al., 2010; Kerfant et al., 2007; Marcantoni et al., 2006). Thus, the lack of contractile effect of the β2-AR in ventricular muscle observed in the present study could be mediated by the inhibition of AC via Gαi and the activation of PDEs induced by PI3K. Beside the role of PI3K on cardiac contractility, the PI3K/PKB signal pathway can induce an anti-apoptotic effect and promote physiological
Fig. 4. Regulation of metabolic and transcriptional signaling by the β2-AR. AMPK (n = 5), PKB (n = 5) and AS160 (n = 5) phosphorylation in the presence of 1 μM CGP20712A alone (CON) or in response to 1 μM tulobuterol (TUL) during 10 min incubation. Each bar represents the average ± S.E.M. Data was analyzed by paired Student t-test. *p b 0.05, **p b 0.01, ***p b 0.001 vs. CON.
896
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897
hypertrophy (Latronico et al., 2004). In fact, PKB is known to prevent apoptosis through its downstream targets GSK3 and CREB and promote cardiomyocyte hypertrophy through its downstream target mTORC1 (Shiojima and Walsh, 2006). Accordingly, in contrast to β1-AR stimulation, which promotes pathological cardiac hypertrophy and cardiomyocyte apoptosis, activation of the β2-AR can exert a cardioprotective effect in a PI3K dependent fashion (Xiao et al., 2004; Yoo et al., 2009). Indeed, β1-agonists are deleterious to patients with heart failure (Persson et al., 1996; The Xamoterol in Severe Heart Failure Study Group, 1990), but the administration of β2-agonists, either alone or combined with β1-antagonists seem to be therapeutically effective (Birks et al., 2006; Schäfers et al., 1994). The mechanisms behind these effects however are not well established. The present results suggest that the cardioprotective effect of β2-AR could potentially be regulated by the activation of the PI3K/PKB signal pathway. The metabolic flexibility of cardiomyocytes is an important factor in the control of heart contractile function. Under basal conditions, when O2 supply and oxidative capacity of cardiomyocytes are not limited, FA metabolism is the primary source of ATP production (Stanley et al., 2005). Conversely, under physiological stress (e.g. exercise) or pathological conditions (e.g. ischemia), there is a switch to CHO metabolism to provide a more immediate source of energy (Stanley et al., 2005). Cardiac metabolism is severely impaired in pathological situations such as cardiac hypertrophy, heart failure and type 2 diabetes, conditions where both CHO and FA oxidation are chronically reduced (van Bilsen et al., 2009). The translocation of the glucose transporter protein 4 (GLUT4) from intracellular vesicles to the plasma membrane is a key step in the regulation of glucose uptake and CHO metabolism. Translocation of GLUT4 is negatively regulated by AS160 (also known as TBC1D4) (Sakamoto and Holman, 2008). The phosphorylation of AS160 at multiple S/T residues promotes its interaction with the 14-3-3 protein, sequestering AS160 and decreasing its inhibitory effect on GLUT4 translocation (Sakamoto and Holman, 2008). The phosphorylation of residue T642 has been shown to be a direct target of both PKB and AMPK (Geraghty et al., 2007). In the heart, PKB and AMPK work together to control glucose uptake (Bertrand et al., 2008). The effects of insulin are mediated by PKB while AMPK mediates the biguanide and exercise effects (Kramer et al., 2006). Moreover, consistent with the increase in p-AMPKαT172 observed in the present study, Li et al. (2010) has recently showed that in rat cardiomyocytes the selective activation of β2-AR increases AMP/ATP ratio and then AMPK activity. A potential mechanism by which β2-AR regulates AMPK is through the activation of PDE by PI3K, regulating thus cAMP and AMP concentration. In fact, an increase in cAMP production or the inhibition of PDEs results in a lower AMPKαT172 phosphorylation and activity (Hurley et al., 2006). Accordingly, Mokni et al. (2010) showed that an increased expression and activity of PDE4 in left ventricle augment the phosphorylation levels of AMPK. These data suggest that the activation of PDEs would activate AMPK by increasing cAMP degradation and the consequent increase in the AMP/ATP ratio. Although this hypothesis needs to be confirmed, the ability of tulobuterol to activate both PKB and AMPK and increase the phosphorylation of AS160 at the critical T642 residue suggests that PKB and AMPK converge on AS160 and could facilitate GLUT4 translocation. To our knowledge, the present study is the first to report that β2-AR stimulation induces the phosphorylation of AMPKαT172 and AS160T642 in rat ventricular muscle. The transcription factor CREB also plays a key role in the control of cardiomyocyte metabolism and plasticity (Hock and Kralli, 2009; Ichiki, 2006). Although CREB can be phosphorylated by PKA (Sands and Palmer, 2008), our data would suggest that it is unlikely that the increase in p-CREBS133 induced by tulobuterol was regulated by PKA, since p-PKAT197 and contractile force did not increased, both factors directly related with PKA activation. However, PKA phosphorylation does not directly reflect its activity, hence, we cannot completely rule out PKA as a potential upstream kinase of CREB under these
experimental conditions. Beside PKA, multiple upstream proteins can phosphorylate CREB on Ser133, including PKB and AMPK (Du and Montminy, 1998; Sands and Palmer, 2008; Thomson et al., 2008). Therefore, CREB activation in response to tulobuterol in the presence of CGP-20712A could be regulated by the activation of PKB and/or AMPK. Prolonged elevation of CREB activity can prevent ventricular cardiomyocyte apoptosis through CREB-induced expression of the antiapoptotic protein Bcl-2 (Ichiki, 2006) and affect metabolism through CREB-dependent regulation of PGC-1α (Wu et al., 2006). Together, these data suggest that the stimulation of cardiac β2-AR might regulate heart performance by the improvement of energetic metabolism of ventricular cardiomyocytes. Conclusion In conclusion, the results of the present study indicate that in rat ventricular muscle, the β2-AR does not exert a direct regulation on ventricular contractility. We demonstrate that selective β2-AR stimulation with tulobuterol activates pathways that are directly involved in the regulation of muscle atrophy/hypertrophy (PKB) and energy metabolism (AMPK), suggesting that long-term β2-AR activation could induce positive adaptations on ventricular function. Therefore, these results are in accordance with the recent evidence indicating a possible beneficial role of β2-AR activation in cardiac muscle (Ahmet et al., 2008; Birks et al., 2006). However, further investigations are still necessary to establish the molecular regulation and clinical relevance of these findings. Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments The authors would like to thank Dr. D. Lee Hamilton for his help and advice with sample preparation and western blot optimization, and Carmen Gonzalez-Muñoz for her technical assistance. Jose Antonio Nadal is gratefully acknowledged for his help with Fig. 2A. References Ahmet I, Krawczyk M, Zhu W, Woo AY, Morrell C, Poosala S, et al. Cardioprotective and survival benefits of long-term combined therapy with β2 adrenoreceptor (AR) agonist and β1 AR blocker in dilated cardiomyopathy postmyocardial infarction. J Pharmacol Exp Ther 2008;325:491–9. Bernstein D, Fajardo G, Zhao M, Urashima T, Powers J, Berry G, et al. Differential cardioprotective/cardiotoxic effects mediated by β-adrenergic receptor subtypes. Am J Physiol Heart Circ Physiol 2005;289:H2441–9. Bertrand L, Horman S, Beauloye C, Vanoverschelde JL. Insulin signaling in the heart. Cardiovasc Res 2008;79:238–48. Birks EJ, Tansley PD, Hardy J, George RS, Bowles CT, Burke M, et al. Left ventricular assist device and drug therapy for the reversal of heart failure. N Engl J Med 2006;355: 1873–84. Communal C, Singh K, Sawyer DB, Colucci WS. Opposing effects of β1- and β2adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxinsensitive G protein. Circulation 1999;100:2210–2. Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem 1998;273:32377–9. Geraghty KM, Chen S, Harthill JE, Ibrahim AF, Toth R, Morrice NA, et al. Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR. Biochem J 2007;407:231–41. Gonzalez-Muñoz C, Fuente T, Hernández-Cascales J. Phosphodiesterases inhibition unmask a positive inotropic effect mediated by β2-adrenoceptors in rat ventricular myocardium. Eur J Pharmacol 2009;607:151–5. Gonzalez-Sicilia L, Perez-Ojeda E, Laorden ML, Brugger AJ, Hernandez J. Tulobuterol: a full β-adrenoceptor agonist or a partial β-agonist with membrane stabilizing activity? Gen Pharmacol 1988;19:103–6. Gregg CJ, Steppan J, Gonzalez DR, Champion HC, Phan AC, Nyhan D, et al. b2-adrenergic receptor-coupled phosphoinositide 3-kinase constrains cAMP-dependent increases in cardiac inotropy through phosphodiesterase 4 activation. Anesth Analg 2010;111:870–7. Hill JA, Olson EN. Cardiac plasticity. N Engl J Med 2008;358:1370–80. Hock MB, Kralli A. Transcriptional control of mitochondrial biogenesis and function. Annu Rev Physiol 2009;71:177–203.
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897 Hurley RL, Barré LK, Wood SD, Anderson KA, Kemp BE, Means AR, et al. Regulation of AMP-activated protein kinase by multisite phosphorylation in response to agents that elevate cellular cAMP. J Biol Chem 2006;281:36662–72. Ichiki T. Role of cAMP response element binding protein in cardiovascular remodeling: good, bad, or both? Arterioscler Thromb Vasc Biol 2006;26:449–55. Jo SH, Leblais V, Wang PH, Crow MT, Xiao RP. Phosphatidylinositol 3-kinase functionally compartmentalizes the concurrent Gs signaling during β2-adrenergic stimulation. Circ Res 2002;91:46–53. Kerfant BG, Zhao D, Lorenzen-Schmidt I, Wilson LS, Cai S, Chen SR, et al. PI3Kγ is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes. Circ Res 2007;101: 400–8. Kramer HF, Witczak CA, Fujii N, Jessen N, Taylor EB, Arnolds DE, et al. Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle. Diabetes 2006;55:2067–76. Kume H. Clinical use of β2-adrenergic receptor agonists based on their intrinsic efficacy. Allergol Int 2005;54:89–97. Laorden ML, Hernández J, Brugger AJ. The beta-agonist drug tulobuterol decreases cardiac automaticity in the rat: comparison with isoproterenol. Gen Pharmacol 1985;16:65–7. Latronico MV, Costinean S, Lavitrano ML, Peschle C, Condorelli G. Regulation of cell size and contractile function by AKT in cardiomyocytes. Ann NY Acad Sci 2004;1015: 250–60. Leblais V, Jo SH, Chakir K, Maltsev V, Zheng M, Crow MT, et al. Phosphatidylinositol 3kinase offsets cAMP-mediated positive inotropic effect via inhibiting Ca2+ influx in cardiomyocytes. Circ Res 2004;95:1183–90. Leroy J, Abi-Gerges A, Nikolaev VO, Richter W, Lechêne P, Mazet JL, et al. Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases. Circ Res 2008;102: 1091–100. Li J, Yan B, Huo Z, Liu Y, Xu J, Sun Y, et al. β2- but not β1-adrenoceptor activation modulates intracellular oxygen availability. J Physiol 2010;588:2987–98. Lynch GS, Ryall JG. Role of β-Adrenoceptor signaling in skeletal muscle: implications for muscle wasting and disease. Physiol Rev 2008;88:729–67. MacNee W. Right heart function in COPD. Semin Respir Crit Care Med 2010;31: 295–312. Marcantoni A, Levi RC, Gallo MP, Hirsch E, Alloatti G. Phosphoinositide 3-kinaseγ (PI3Kγ) controls L-type calcium current (ICa, L) through its positive modulation of type-3 phosphodiesterase (PDE3). J Cell Physiol 2006;206:329–36. McConville P, Spencer RG, Lakatta EG. Temporal dynamics of inotropic, chronotropic and metabolic responses during β1- and β2-AR stimulation in the isolated, perfused rat heart. Am J Physiol Endocrinol Metab 2005;289:E412–8. Mokni W, Keravis T, Etienne-Selloum N, Walter A, Kane MO, Schini-Kerth VB, et al. Concerted regulation of cGMP and cAMP phosphodiesterases in early cardiac hypertrophy induced by angiotensin II. PLoS One 2010;5:e14227. Morin D, Sapena R, Tillement JP, Urien S. Evidence for different interactions between β1- and β2-adrenoceptor subtypes with adenylyl cyclase in the rat brain: a concentration–response study using forskolin. Pharmacol Res 2000;41:435–43. Nevzorova J, Evans BA, Bengtsson T, Summers RJ. Multiple signalling pathways involved in β2-adrenoceptor-mediated glucose uptake in rat skeletal muscle cells. Br J Pharmacol 2006;147:446–54. Ngala RA, O'Dowd J, Wang SJ, Agarwal A, Stocker C, Cawthorne MA, et al. Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and β2-adrenoceptor mechanisms. Br J Pharmacol 2008;155:395–406. Patel KR. Tulobuterol in the management of obstructive airways disease in adults. Clin Ther 1985;7:452–67. Persson H, Eriksson SV, Erhardt L. Effects of beta receptor antagonists on left ventricular function in patients with clinical evidence of heart failure after myocardial infarction. A double-blind comparison of metoprolol and xamoterol. Echocardio-
897
graphic results from the Metoprolol and Xamoterol Infarction Study (MEXIS). Eur Heart J 1996;17:741–9. Pönicke K, Heinroth-Hoffmann I, Brodde OE. Role of β1- and β2-adrenoceptors in hypertrophic and apoptotic effects of noradrenaline and adrenaline in adult rat ventricular cardiomyocytes. Naunyn Schmiedebergs Arch Pharmacol 2003;367: 592–9. Rochais F, Abi-Gerges A, Horner K, Lefebvre F, Cooper DM, Conti M, et al. A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gscoupled receptors in adult rat ventricular myocytes. Circ Res 2006;98:1081–8. Ruff F, Zander JF, Edoute Y, Santais MC, Flavahan NA, Verbeuren TJ, et al. Beta-2 adrenergic responses to tulobuterol in airway smooth muscle, vascular smooth muscle and adrenergic nerves. J Pharmacol Exp Ther 1988;244:173–80. Sakamoto K, Holman GD. Emerging role for AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic. Am J Physiol Endocrinol Metab 2008;295:E29–37. Salazar NC, Chen J, Rockman HA. Cardiac GPCRs: GPCR signaling in healthy and failing hearts. Biochim Biophys Acta 2007;1768:1006–18. Sands WA, Palmer TM. Regulating gene transcription in response to cyclic AMP elevation. Cell Signal 2008;20:460–6. Schäfers RF, Adler S, Daul A, Zeitler G, Vogelsang M, Zerkowski HR, et al. Positive inotropic effects of the β2-adrenoceptor agonist terbutaline in the human heart: effects of long-term β1-adrenoceptor antagonist treatment. J Am Coll Cardiol 1994;23:1224–33. Shiojima I, Walsh K. Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway. Genes Dev 2006;20:3347–65. Siedlecka U, Arora M, Kolettis T, Soppa GK, Lee J, Stagg MA, et al. Effects of clenbuterol on contractility and Ca2+ homeostasis of isolated rat ventricular myocytes. Am J Physiol Heart Circ Physiol 2008;295:H1917–26. Soppa GK, Smolenski RT, Latif N, Yuen AH, Malik A, Karbowska J, et al. Effects of chronic administration of clenbuterol on function and metabolism of adult rat cardiac muscle. Am J Physiol Heart Circ Physiol 2005;288:H1468–76. Stanley WC, Recchia FA, Lopaschuk GD. Myocardial substrate metabolism in the normal and failing heart. Physiol Rev 2005;85:1093–129. The Xamoterol in Severe Heart Failure Study Group. Xamoterol in severe heart failure. Lancet 1990;336:1–6. Terpstra GK, Raaijmakers JA. Beta-agonistic properties of tulobuterol, a new betasympathicomimetic drug, and its effects on pulmonary beta-adrenoceptor characteristics. Lung 1990;168:179–85. Thomson DM, Herway ST, Fillmore N, Kim H, Brown JD, Barrow JR, et al. AMP-activated protein kinase phosphorylates transcription factors of the CREB family. J Appl Physiol 2008;104:429–38. van Bilsen M, van Nieuwenhoven FA, van der Vusse GJ. Metabolic remodelling of the failing heart: beneficial or detrimental? Cardiovasc Res 2009;81:420–8. Wu Z, Huang X, Feng Y, Handschin C, Feng Y, Gullicksen PS, et al. Transducer of regulated CREB-binding proteins (TORCs) induce PGC-1α transcription and mitochondrial biogenesis in muscle cells. Proc Natl Acad Sci U S A 2006;103:14379–84. Xiao RP, Ji X, Lakatta EG. Functional coupling of the β2-adrenoceptor to a pertussis toxin-sensitive G protein in cardiac myocytes. Mol Pharmacol 1995;47:322–9. Xiao RP, Zhu W, Zheng M, Chakir K, Bond R, Lakatta EG, et al. Subtype-specific βadrenoceptor signaling pathways in the heart and their potential clinical implications. Trends Pharmacol Sci 2004;25:358–65. Yamamoto DL, Hutchinson DS, Bengtsson T. β2-adrenergic activation increases glycogen synthesis in L6 skeletal muscle cells through a signaling pathway independent of cyclic AMP. Diabetologia 2007;50:158–67. Yoo B, Lemaire A, Mangmool S, Wolf MJ, Curcio A, Mao L, et al. β1-adrenergic receptors stimulate cardiac contractility and CaMKII activation in vivo and enhance cardiac dysfunction following myocardial infarction. Am J Physiol Heart Circ Physiol 2009;297:H1377–86. Zhu WZ, Zheng M, Koch WJ, Lefkowitz RJ, Kobilka BK, Xiao RP. Dual modulation of cell survival and cell death by β2-adrenergic signaling in adult mouse cardiac myocytes. Proc Natl Acad Sci U S A 2001;98:1607–12.
Contents lists available at ScienceDirect
Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle Joaquín Pérez-Schindler a,⁎, Andrew Philp b,1, Keith Baar b,1, Jesús Hernández-Cascales a a b
Department of Pharmacology, Faculty of Medicine, University of Murcia, Murcia, Spain Division of Molecular Physiology, University of Dundee, Dundee, UK
a r t i c l e
i n f o
Article history: Received 7 September 2010 Accepted 25 March 2011 Keywords: β2-adrenergic receptor Cardiac contractility Cardiac metabolism AMPK PI3K
a b s t r a c t Aims: Cardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (βAR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β2-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β2-AR activation by tulobuterol. Main methods: Strips of rat right ventricle were electrically stimulated (1 Hz) in standard Tyrode solution (95% O2, 5% CO2) in the presence of the β1-antagonist CGP-20712A (1 μM). A cumulative dose–response curve for tulobuterol (0.1–10 μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30 μM), or 10 min incubation (1 μM) with the β2-agonist tulobuterol was performed. Key findings: β2-AR stimulation induced a positive inotropic effect (maximal effect = 33 ± 3.3%) and a decrease in the time required for half relaxation (from 45 ± 0.6 to 31 ± 1.8 ms, − 30%, p b 0.001) after the inhibition of PDEs. After 10 min of β2-AR stimulation, p-AMPKαT172 (54%), p-PKBT308 (38%), p-AS160T642 (46%) and p-CREBS133 (63%) increased, without any change in p-PKAT197. Significance: These results suggest that the regulation of ventricular contractility is not the primary function of the β2-AR. Rather, β2-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle. © 2011 Elsevier Inc. All rights reserved.
Introduction The contractile and metabolic proprieties of cardiac muscle are critical factors in the regulation of heart performance and the susceptibility to cardiac dysfunction (Hill and Olson, 2008). Heart function is modulated by the sympathetic nervous system through βadrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. In the heart, the β1 and β2-AR are the main receptors expressed (Salazar et al., 2007) and are each thought to activate alternate signaling pathways and regulate diverse cellular responses (Xiao et al., 2004). Stimulation of the β2-AR exerts a cardioprotective effect, whereas β1-AR stimulation has a negative effect on cardiac function (Bernstein et al., 2005; Xiao et al., 2004; Zhu et al., 2001). The β1-AR is the most abundant subtype in the heart and the main regulator of cardiac contractility (Salazar et al., 2007; Yoo et al., 2009). β1-AR modulates cardiac function via the stimulatory G protein (Gαs)/
⁎ Corresponding author at: Focal Area Growth and Development, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland. Tel.: + 41 61 267 2237; fax: + 41 61 267 2221. E-mail address: [email protected] (J. Pérez-Schindler). 1 Current address: Neurobiology, Physiology and Behavior, University of California, Davis, CA, United States. 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.03.020
adenylyl cyclase (AC)/cyclic AMP (cAMP)/cAMP dependent protein kinase (PKA) signal pathway, leading to the phosphorylation of proteins involved in regulating excitation-contraction coupling in cardiac muscle (Salazar et al., 2007). β2-AR is coupled not only to Gαs but to an inhibitory G protein (Gαi), that can block AC and prevent PKA activation (Xiao et al., 1995). This dual coupling to Gαs and Gαi is consistent with the lower inotropic response reported in rat heart following administration of β2 compared to β1-AR agonists (McConville et al., 2005). Therefore, the regulation of cardiac muscle contractility does not seem to be the primary function of β2-AR in rodent heart. In addition to inhibiting Gαs/AC/PKA, Gαi has other functions within the heart. Together with the Gβγ heterodimer, Gαi can activate phosphatidylinositol 3-kinase (PI3K) (Jo et al., 2002). In fact, some β2agonists induce hypertrophy of cardiac and skeletal muscle (Lynch and Ryall, 2008; Pönicke et al., 2003) and prevent apoptosis in a Gαi/ PI3K dependent manner (Communal et al., 1999; Zhu et al., 2001). In association with the increase in cardiac muscle mass, β2agonists also affect muscle metabolism. Soppa et al. (2005) demonstrated that chronic treatment with the β2-agonist clenbuterol increased carbohydrate (CHO) contribution to the tricarboxylic acid (TCA) cycle in rat ventricular cardiomyocytes, whereas the stimulation of β2-AR in skeletal muscle increases fatty acid (FA) oxidation while glucose uptake is diverted towards glycogen synthesis (Nevzorova et al., 2006; Ngala et al., 2008; Yamamoto et al., 2007).
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897
Collectively, these studies provide evidence that the β2-AR could play an important role in the regulation of cardiac metabolism, however, to date, the cellular signaling pathways involved in these responses are not well characterized. Chronic obstructive pulmonary disease (COPD) is associated with the development of pulmonary hypertension, which impairs right ventricle function by inducing pathological hypertrophy (MacNee, 2010). Therefore, the assessment of the impact of β2-agonists on right ventricle function has a particular interest for the treatment of COPD. Tulobuterol is a β2-agonists used for long-term treatment of COPD in humans (Patel, 1985). Besides early pharmacological studies (GonzalezSicilia et al., 1988; Laorden et al., 1985; Ruff et al., 1988), the effects of tulobuterol on cardiac function have not been examined. Therefore, the aim of the present study was to determinate the effect of selective β2-AR stimulation with tulobuterol on contractility and intracellular signaling in rat right ventricle preparations ex vivo.
893
single dose of 1 μM tulobuterol for 10 min. On completion, cardiac strips were frozen in liquid nitrogen and stored at − 80 °C until analysis. Protein isolation Samples were pulverized on dry ice, homogenized with a polytron in 200 μL of ice cold sucrose lysis buffer (50 mM Tris pH 7.5, 250 mM Sucrose, 1 mM EDTA, 1 mM EGTA, 1% Triton X 100, 50 mM NaF, 1 mM Na3VO4, 50 mM Na4P2O7, 0.1% DTT) and shaken for 30 min at 4 °C. Samples were then centrifuged at 14 000 rpm for 10 min at 4 °C and the protein concentration of the supernatant determined by the DC protein assay (Bio-Rad, Hercules CA). Equal aliquots of protein were boiled for 5 min in Laemmli sample buffer (250 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 5% β-mercaptoetanol). Western blot
Materials and methods The study was performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and approved by the Ethical Committee of the University of Murcia. Animals and right ventricle isolation Male adult Sprague–Dawley rats (250–300 g) were killed by a blow to the head followed by cervical dislocation. The heart was rapidly removed and placed in Tyrode solution (136.9 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1.5 mM MgCl2, 0.4 mM NaH2PO4, 11.9 mM NaHCO3, 5 mM D-glucose and 5 mM pyruvate) maintained at pH 7.4 and gassed with 95% O2 and 5% CO2. The external wall of the right ventricle was extracted and cut into strips ~1 mm wide, ~10 mm long and ~ 0.5 mm thick. Subsequently, the strips were mounted longitudinally between two platinum electrodes, placed in an organ bath (30 mL) with Tyrode solution (37 °C, pH 7.4, 95% O2 and 5% CO2) and electrically stimulated (Grass SD-9) against a preload of ~ 1 g at 1 Hz, 5 ms and supramaximal voltage (~ 25% above response threshold). Contraction amplitude and relaxation was measured with a force transducer (Grass FT-03) and amplified (Stemtech Inc., Houston, Texas) for future quantification. The strips were allowed to equilibrate with the experimental conditions for ~40 min before drug treatment. Cumulative dose–response curve After equilibration, a cumulative dose–response curve with the β2agonist tulobuterol (0.1–10 μM; Lab. Ferrer, Co. Spain) in the presence of the β1-antagonist CGP-20712A (1 μM; Sigma) was performed. To determinate the selective β2-AR activation, the β2-antagonist ICI118,551 (50 nM; Sigma) was used. The role of the cyclic nucleotide phosphodiesterase (PDE) was determined using the non selective PDE inhibitor IBMX (30 μM; Sigma). The β-antagonists and IBMX were added to the bath 15 min before tulobuterol addition. Drugs were added to the organ bath (30 mL) in a volume no higher than 0.1 mL. The experiments were finalized with the addition of 9 mM CaCl2 to determinate the response capacity and maximal contractile amplitude of the preparations. Changes in contractile force induced by tulobuterol are expressed as a percentage of the control contraction amplitude (in the presence of the corresponding β-antagonist and IBMX, if applicable). The relaxation capacity of the strips was studied by the determination of the time required for half relaxation (t1/2). Incubation for protein phosphorylation measurement To determine the metabolic signaling regulated in response to the selective β2-AR activation, the strips were incubated after the equilibration with 1 μM CGP-20712A alone or in combination with a
Samples were separated on an SDS-polyacrylamide gel (8.25%) for 50 min and then transferred to a Protran nitrocellulose membrane (Whatman, Dassel, Germany) for 60 min. Membranes were blocked for 1 h in 3% milk in Tris-buffered saline +0.1% tween-20 (TBST) before overnight incubation at 4 °C with appropriate primary antibody in TBST (1:1000 dilution). Proteins were detected with a primary antibody to p-PKBT308 (Cell Signaling; 4056), p-AMPKαT172 (Cell Signaling; 2535), p-CREBS133 (Rockland; 600-401-270), p-PKAT197 (Cell Signaling; 4781), the primary antibodies to p-AS160T642 were kindly provided by Professor Grahame Hardie (Division of Molecular Physiology, University of Dundee). Since the tulobuterol treatments lasted only 10 min and therefore the total amount of the signaling proteins was unlikely to change, eEF2 (Cell Signaling; 2332) was used as a loading control for all western blots. Following incubation, membranes were washed 3 times with TBST before incubation with an appropriate peroxidase-conjugated secondary antibody in TBST (1:10 000 dilution; Pierce, Rockford, IL). Antibody binding was detected using enhanced chemiluminescence HRP substrate detection kit (Millipore, Billerica, MA). Imaging and band quantification were carried out using a ChemiGenius Bioimaging Gel Doc System (Syngene, Cambridge, UK). Statistical analysis All values are expressed as mean ± S.E.M (n = 4–5 independent experiments). The log EC50 was calculated by the analysis of the non linear regression of the dose–response curve (Graph Pad Software, San Diego, CA). The statistical significance was determined using the Student t-test for paired and unpaired data. The significance difference was set at p b 0.05. Results Inotropic and lusitropic effect of the selective β2-AR stimulation The β-antagonists and IBMX did not alter contraction amplitude compared to basal levels (data not shown). In addition to its effects on β2-AR stimulation, tulobuterol has also been suggested to stimulate β1-AR at high concentrations (Morin et al., 2000). To ensure that the effects of tulobuterol were β2-AR specific, the β1-antagonist CGP20712A was used in combination with tulobuterol. Thus, tulobuterol in the presence of CGP-20712A did not produce a positive inotropic effect (n = 4; Fig. 1A and B). This data provides preliminary evidence that activation of the β2-AR does not increase the contractile force of the heart. However, IBMX in combination with CGP-20712A produced a dose-dependent positive inotropic effect in response to tulobuterol, with a maximal effect of 33 ± 3.3% and a log EC50: − 6.9 ± 0.6 M (n = 5; Fig. 1A and B). Moreover, under these conditions, tulobuterol not only increased the contractile force but also induced a decrease in
894
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897
by the β2-antagonist ICI-118,551 (n = 4; Fig. 1A and B), suggesting that the β2-AR is required for positive inotropy under these experimental conditions. PKA and CREB regulation by the β2-AR To induce selective β2-AR stimulation and explore the intracellular signaling mediated by this receptor, ventricular strips were incubated with the β1-antagonist CGP-20712A alone or in the presence of tulobuterol. After ten minutes of selective β2-AR stimulation with tulobuterol, there was no change (p = 0.09) in the phosphorylation of PKAT197 (n= 5; Fig. 3). In contrast to its effects on PKA, tulobuterol induced a significant increase (63%; n = 4, p b 0.01) in the phosphorylation of CREBS133 (Fig. 3). Metabolic signaling induced by the β2-AR Tulobuterol in the presence of CGP-20712A increased the phosphorylation of AMPKαT172 by 54% (n = 5, p b 0.001; Fig. 4). Additionally, 10 min of incubation with tulobuterol in the presence of
Fig. 1. Inotropic response to the β2-agonist tulobuterol. Representative experiment (A) and average values (B) of the modification of contractile force in response to tulobuterol (0.1-10 μM) in the presence of 1 μM CGP-20712A alone (n= 4, CGP20) or in combination with 30 μM IBMX (n= 5, CGP20 + IBMX) or in addition to 50 nM ICI-118,551 (n= 4, CGP20 + IBMX + ICI). Each point represents the average ±S.E.M. Data was analyzed by unpaired Student t-test. **p b 0.01, ***p b 0.001 vs. CGP20 and CGP20 + IBMX+ ICI.
the t1/2 from 45 ± 0.6 to 31 ± 1.8 ms (n = 5, − 30%, p b 0.001; Fig. 2) suggesting that PDE need to be inhibited before β2-agonists increase inotropy. In support of this, positive inotropy was completely blocked
Fig. 2. Lusitropic response to the β2-agonist tulobuterol. Representative experiment (A) and average values (B) of the decrease in the time required to the half relaxation time (t1/2) in the presence of 1 μM CGP-20712A combined with 30 μM IBMX alone (n= 5, CON) or in response to 3 μM tulobuterol (n= 5, TUL). Each bar represents the average±S.E.M. Data was analyzed by paired Student t-test. ***p b 0.001 vs. CON.
Fig. 3. Regulation of downstream proteins of the β2-AR. PKA (n = 5) and CREB (n = 4) phosphorylation in the presence of 1 μM CGP-20712A alone (CON) or in response to 1 μM tulobuterol (TUL) during 10 min incubation. Each bar represents the average ±S.E.M. Data was analyzed by paired Student t-test. **p b 0.01 vs. CON.
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897
CGP-20712A resulted in an increase in the phosphorylation of PKBT308 (n = 5, 38%, p b 0.05; Fig. 4). Along with the changes in AMPK and PKB phosphorylation, the phosphorylation of their downstream target
895
AS160T642 was significantly increased in the tulobuterol treated strips (n = 5, 46%, p b 0.01; Fig. 4). The phosphorylation of p38T180/Y182 decreased − 8.6% (data not shown) in response to the selective activation of β2-AR with tulobuterol, suggesting that the p38 MAPK pathway is not a target for Tulobuterol. Discussion The results obtained in the present study demonstrate that the β2AR directly regulates metabolic signaling, however the β2-AR does not appear to modulate contractility in rat ventricular muscle without concomitant inhibition of PDE. Selective β2-AR stimulation with tulobuterol did not enhance the contractile force of ventricular strips, but did produce an increase in the phosphorylation of AMPKαT172, PKBT308 and their downstream targets AS160T642 and the transcription factor CREBS133, suggesting a direct link between β2-AR and intracellular signaling associated with the modulation of energetic metabolism and cardiac muscle plasticity. The β2-agonist tulobuterol is a bronchodilator characterized by an EC50 of 0.15 μM, an intrinsic efficacy of 35% of isoproterenol and a halflife of 3.1 h (Kume, 2005; Patel, 1985; Terpstra and Raaijmakers, 1990). While tulobuterol weakly stimulate β1-AR in rat brain (Morin et al., 2000), in the heart it has a minimal effect on β1-AR and cardiac contractility (Gonzalez-Sicilia et al., 1988; Laorden et al., 1985; Ruff et al., 1988). The absence of a positive inotropic effect in response to selective β2-AR stimulation with tulobuterol is consistent with previous reports that show that the positive inotropic effect of β2agonists (e.g. fenoterol) can be blocked by CGP-20712A (GonzalezMuñoz et al., 2009; Siedlecka et al., 2008). This would suggest that the variability in the inotropic effect induced by different β2-agonists is, at least in part, mediated by the activation of the β1-AR. Therefore, given the deleterious effects of β1-AR stimulation on heart function, tulobuterol could be a more attractive therapeutic drug in comparison with other β2-agonists. The contractile effect of selective β2-AR stimulation was only observed when PDEs (Fig. 1) or Gαi (Gonzalez-Muñoz et al., 2009) were inhibited, resulting in positive lusitropy and a dose-dependent inotropic effect. This suggests that the contractile effects of β2-AR stimulation are dependent on cAMP production and degradation and provide evidence of a critical role for PDEs in modulating these responses. However, both Gαi and PDEs are functionally active in normal cardiomyocytes and induce a negative effect on Gαs signaling and this may explain why contractile force and PKA phosphorylation did not increase in response to selective β2-AR activation with tulobuterol. Tulobuterol in the presence of the CGP-20712A increased the phosphorylation of PKBT308, which is a key downstream protein in the PI3K signaling pathway (Latronico et al., 2004), further supporting a role for PI3K in the β2-AR signaling. In ventricular cardiomyocytes, PI3K can blunt the inotropic response to both β1 and β2-AR stimulation (Jo et al., 2002; Leblais et al., 2004). The possible mechanism by which PI3K exerts this effect on contractility could be through the activation of PDEs and the resultant decrease in cAMP (Gregg et al., 2010). Indeed, β-ARs are functionally coupled to PDEs (Rochais et al., 2006) and PDEs can be activated by β-AR (Leroy et al., 2008) and PI3K (Gregg et al., 2010; Kerfant et al., 2007; Marcantoni et al., 2006). Thus, the lack of contractile effect of the β2-AR in ventricular muscle observed in the present study could be mediated by the inhibition of AC via Gαi and the activation of PDEs induced by PI3K. Beside the role of PI3K on cardiac contractility, the PI3K/PKB signal pathway can induce an anti-apoptotic effect and promote physiological
Fig. 4. Regulation of metabolic and transcriptional signaling by the β2-AR. AMPK (n = 5), PKB (n = 5) and AS160 (n = 5) phosphorylation in the presence of 1 μM CGP20712A alone (CON) or in response to 1 μM tulobuterol (TUL) during 10 min incubation. Each bar represents the average ± S.E.M. Data was analyzed by paired Student t-test. *p b 0.05, **p b 0.01, ***p b 0.001 vs. CON.
896
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897
hypertrophy (Latronico et al., 2004). In fact, PKB is known to prevent apoptosis through its downstream targets GSK3 and CREB and promote cardiomyocyte hypertrophy through its downstream target mTORC1 (Shiojima and Walsh, 2006). Accordingly, in contrast to β1-AR stimulation, which promotes pathological cardiac hypertrophy and cardiomyocyte apoptosis, activation of the β2-AR can exert a cardioprotective effect in a PI3K dependent fashion (Xiao et al., 2004; Yoo et al., 2009). Indeed, β1-agonists are deleterious to patients with heart failure (Persson et al., 1996; The Xamoterol in Severe Heart Failure Study Group, 1990), but the administration of β2-agonists, either alone or combined with β1-antagonists seem to be therapeutically effective (Birks et al., 2006; Schäfers et al., 1994). The mechanisms behind these effects however are not well established. The present results suggest that the cardioprotective effect of β2-AR could potentially be regulated by the activation of the PI3K/PKB signal pathway. The metabolic flexibility of cardiomyocytes is an important factor in the control of heart contractile function. Under basal conditions, when O2 supply and oxidative capacity of cardiomyocytes are not limited, FA metabolism is the primary source of ATP production (Stanley et al., 2005). Conversely, under physiological stress (e.g. exercise) or pathological conditions (e.g. ischemia), there is a switch to CHO metabolism to provide a more immediate source of energy (Stanley et al., 2005). Cardiac metabolism is severely impaired in pathological situations such as cardiac hypertrophy, heart failure and type 2 diabetes, conditions where both CHO and FA oxidation are chronically reduced (van Bilsen et al., 2009). The translocation of the glucose transporter protein 4 (GLUT4) from intracellular vesicles to the plasma membrane is a key step in the regulation of glucose uptake and CHO metabolism. Translocation of GLUT4 is negatively regulated by AS160 (also known as TBC1D4) (Sakamoto and Holman, 2008). The phosphorylation of AS160 at multiple S/T residues promotes its interaction with the 14-3-3 protein, sequestering AS160 and decreasing its inhibitory effect on GLUT4 translocation (Sakamoto and Holman, 2008). The phosphorylation of residue T642 has been shown to be a direct target of both PKB and AMPK (Geraghty et al., 2007). In the heart, PKB and AMPK work together to control glucose uptake (Bertrand et al., 2008). The effects of insulin are mediated by PKB while AMPK mediates the biguanide and exercise effects (Kramer et al., 2006). Moreover, consistent with the increase in p-AMPKαT172 observed in the present study, Li et al. (2010) has recently showed that in rat cardiomyocytes the selective activation of β2-AR increases AMP/ATP ratio and then AMPK activity. A potential mechanism by which β2-AR regulates AMPK is through the activation of PDE by PI3K, regulating thus cAMP and AMP concentration. In fact, an increase in cAMP production or the inhibition of PDEs results in a lower AMPKαT172 phosphorylation and activity (Hurley et al., 2006). Accordingly, Mokni et al. (2010) showed that an increased expression and activity of PDE4 in left ventricle augment the phosphorylation levels of AMPK. These data suggest that the activation of PDEs would activate AMPK by increasing cAMP degradation and the consequent increase in the AMP/ATP ratio. Although this hypothesis needs to be confirmed, the ability of tulobuterol to activate both PKB and AMPK and increase the phosphorylation of AS160 at the critical T642 residue suggests that PKB and AMPK converge on AS160 and could facilitate GLUT4 translocation. To our knowledge, the present study is the first to report that β2-AR stimulation induces the phosphorylation of AMPKαT172 and AS160T642 in rat ventricular muscle. The transcription factor CREB also plays a key role in the control of cardiomyocyte metabolism and plasticity (Hock and Kralli, 2009; Ichiki, 2006). Although CREB can be phosphorylated by PKA (Sands and Palmer, 2008), our data would suggest that it is unlikely that the increase in p-CREBS133 induced by tulobuterol was regulated by PKA, since p-PKAT197 and contractile force did not increased, both factors directly related with PKA activation. However, PKA phosphorylation does not directly reflect its activity, hence, we cannot completely rule out PKA as a potential upstream kinase of CREB under these
experimental conditions. Beside PKA, multiple upstream proteins can phosphorylate CREB on Ser133, including PKB and AMPK (Du and Montminy, 1998; Sands and Palmer, 2008; Thomson et al., 2008). Therefore, CREB activation in response to tulobuterol in the presence of CGP-20712A could be regulated by the activation of PKB and/or AMPK. Prolonged elevation of CREB activity can prevent ventricular cardiomyocyte apoptosis through CREB-induced expression of the antiapoptotic protein Bcl-2 (Ichiki, 2006) and affect metabolism through CREB-dependent regulation of PGC-1α (Wu et al., 2006). Together, these data suggest that the stimulation of cardiac β2-AR might regulate heart performance by the improvement of energetic metabolism of ventricular cardiomyocytes. Conclusion In conclusion, the results of the present study indicate that in rat ventricular muscle, the β2-AR does not exert a direct regulation on ventricular contractility. We demonstrate that selective β2-AR stimulation with tulobuterol activates pathways that are directly involved in the regulation of muscle atrophy/hypertrophy (PKB) and energy metabolism (AMPK), suggesting that long-term β2-AR activation could induce positive adaptations on ventricular function. Therefore, these results are in accordance with the recent evidence indicating a possible beneficial role of β2-AR activation in cardiac muscle (Ahmet et al., 2008; Birks et al., 2006). However, further investigations are still necessary to establish the molecular regulation and clinical relevance of these findings. Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments The authors would like to thank Dr. D. Lee Hamilton for his help and advice with sample preparation and western blot optimization, and Carmen Gonzalez-Muñoz for her technical assistance. Jose Antonio Nadal is gratefully acknowledged for his help with Fig. 2A. References Ahmet I, Krawczyk M, Zhu W, Woo AY, Morrell C, Poosala S, et al. Cardioprotective and survival benefits of long-term combined therapy with β2 adrenoreceptor (AR) agonist and β1 AR blocker in dilated cardiomyopathy postmyocardial infarction. J Pharmacol Exp Ther 2008;325:491–9. Bernstein D, Fajardo G, Zhao M, Urashima T, Powers J, Berry G, et al. Differential cardioprotective/cardiotoxic effects mediated by β-adrenergic receptor subtypes. Am J Physiol Heart Circ Physiol 2005;289:H2441–9. Bertrand L, Horman S, Beauloye C, Vanoverschelde JL. Insulin signaling in the heart. Cardiovasc Res 2008;79:238–48. Birks EJ, Tansley PD, Hardy J, George RS, Bowles CT, Burke M, et al. Left ventricular assist device and drug therapy for the reversal of heart failure. N Engl J Med 2006;355: 1873–84. Communal C, Singh K, Sawyer DB, Colucci WS. Opposing effects of β1- and β2adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxinsensitive G protein. Circulation 1999;100:2210–2. Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem 1998;273:32377–9. Geraghty KM, Chen S, Harthill JE, Ibrahim AF, Toth R, Morrice NA, et al. Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR. Biochem J 2007;407:231–41. Gonzalez-Muñoz C, Fuente T, Hernández-Cascales J. Phosphodiesterases inhibition unmask a positive inotropic effect mediated by β2-adrenoceptors in rat ventricular myocardium. Eur J Pharmacol 2009;607:151–5. Gonzalez-Sicilia L, Perez-Ojeda E, Laorden ML, Brugger AJ, Hernandez J. Tulobuterol: a full β-adrenoceptor agonist or a partial β-agonist with membrane stabilizing activity? Gen Pharmacol 1988;19:103–6. Gregg CJ, Steppan J, Gonzalez DR, Champion HC, Phan AC, Nyhan D, et al. b2-adrenergic receptor-coupled phosphoinositide 3-kinase constrains cAMP-dependent increases in cardiac inotropy through phosphodiesterase 4 activation. Anesth Analg 2010;111:870–7. Hill JA, Olson EN. Cardiac plasticity. N Engl J Med 2008;358:1370–80. Hock MB, Kralli A. Transcriptional control of mitochondrial biogenesis and function. Annu Rev Physiol 2009;71:177–203.
J. Pérez-Schindler et al. / Life Sciences 88 (2011) 892–897 Hurley RL, Barré LK, Wood SD, Anderson KA, Kemp BE, Means AR, et al. Regulation of AMP-activated protein kinase by multisite phosphorylation in response to agents that elevate cellular cAMP. J Biol Chem 2006;281:36662–72. Ichiki T. Role of cAMP response element binding protein in cardiovascular remodeling: good, bad, or both? Arterioscler Thromb Vasc Biol 2006;26:449–55. Jo SH, Leblais V, Wang PH, Crow MT, Xiao RP. Phosphatidylinositol 3-kinase functionally compartmentalizes the concurrent Gs signaling during β2-adrenergic stimulation. Circ Res 2002;91:46–53. Kerfant BG, Zhao D, Lorenzen-Schmidt I, Wilson LS, Cai S, Chen SR, et al. PI3Kγ is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes. Circ Res 2007;101: 400–8. Kramer HF, Witczak CA, Fujii N, Jessen N, Taylor EB, Arnolds DE, et al. Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle. Diabetes 2006;55:2067–76. Kume H. Clinical use of β2-adrenergic receptor agonists based on their intrinsic efficacy. Allergol Int 2005;54:89–97. Laorden ML, Hernández J, Brugger AJ. The beta-agonist drug tulobuterol decreases cardiac automaticity in the rat: comparison with isoproterenol. Gen Pharmacol 1985;16:65–7. Latronico MV, Costinean S, Lavitrano ML, Peschle C, Condorelli G. Regulation of cell size and contractile function by AKT in cardiomyocytes. Ann NY Acad Sci 2004;1015: 250–60. Leblais V, Jo SH, Chakir K, Maltsev V, Zheng M, Crow MT, et al. Phosphatidylinositol 3kinase offsets cAMP-mediated positive inotropic effect via inhibiting Ca2+ influx in cardiomyocytes. Circ Res 2004;95:1183–90. Leroy J, Abi-Gerges A, Nikolaev VO, Richter W, Lechêne P, Mazet JL, et al. Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases. Circ Res 2008;102: 1091–100. Li J, Yan B, Huo Z, Liu Y, Xu J, Sun Y, et al. β2- but not β1-adrenoceptor activation modulates intracellular oxygen availability. J Physiol 2010;588:2987–98. Lynch GS, Ryall JG. Role of β-Adrenoceptor signaling in skeletal muscle: implications for muscle wasting and disease. Physiol Rev 2008;88:729–67. MacNee W. Right heart function in COPD. Semin Respir Crit Care Med 2010;31: 295–312. Marcantoni A, Levi RC, Gallo MP, Hirsch E, Alloatti G. Phosphoinositide 3-kinaseγ (PI3Kγ) controls L-type calcium current (ICa, L) through its positive modulation of type-3 phosphodiesterase (PDE3). J Cell Physiol 2006;206:329–36. McConville P, Spencer RG, Lakatta EG. Temporal dynamics of inotropic, chronotropic and metabolic responses during β1- and β2-AR stimulation in the isolated, perfused rat heart. Am J Physiol Endocrinol Metab 2005;289:E412–8. Mokni W, Keravis T, Etienne-Selloum N, Walter A, Kane MO, Schini-Kerth VB, et al. Concerted regulation of cGMP and cAMP phosphodiesterases in early cardiac hypertrophy induced by angiotensin II. PLoS One 2010;5:e14227. Morin D, Sapena R, Tillement JP, Urien S. Evidence for different interactions between β1- and β2-adrenoceptor subtypes with adenylyl cyclase in the rat brain: a concentration–response study using forskolin. Pharmacol Res 2000;41:435–43. Nevzorova J, Evans BA, Bengtsson T, Summers RJ. Multiple signalling pathways involved in β2-adrenoceptor-mediated glucose uptake in rat skeletal muscle cells. Br J Pharmacol 2006;147:446–54. Ngala RA, O'Dowd J, Wang SJ, Agarwal A, Stocker C, Cawthorne MA, et al. Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and β2-adrenoceptor mechanisms. Br J Pharmacol 2008;155:395–406. Patel KR. Tulobuterol in the management of obstructive airways disease in adults. Clin Ther 1985;7:452–67. Persson H, Eriksson SV, Erhardt L. Effects of beta receptor antagonists on left ventricular function in patients with clinical evidence of heart failure after myocardial infarction. A double-blind comparison of metoprolol and xamoterol. Echocardio-
897
graphic results from the Metoprolol and Xamoterol Infarction Study (MEXIS). Eur Heart J 1996;17:741–9. Pönicke K, Heinroth-Hoffmann I, Brodde OE. Role of β1- and β2-adrenoceptors in hypertrophic and apoptotic effects of noradrenaline and adrenaline in adult rat ventricular cardiomyocytes. Naunyn Schmiedebergs Arch Pharmacol 2003;367: 592–9. Rochais F, Abi-Gerges A, Horner K, Lefebvre F, Cooper DM, Conti M, et al. A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gscoupled receptors in adult rat ventricular myocytes. Circ Res 2006;98:1081–8. Ruff F, Zander JF, Edoute Y, Santais MC, Flavahan NA, Verbeuren TJ, et al. Beta-2 adrenergic responses to tulobuterol in airway smooth muscle, vascular smooth muscle and adrenergic nerves. J Pharmacol Exp Ther 1988;244:173–80. Sakamoto K, Holman GD. Emerging role for AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic. Am J Physiol Endocrinol Metab 2008;295:E29–37. Salazar NC, Chen J, Rockman HA. Cardiac GPCRs: GPCR signaling in healthy and failing hearts. Biochim Biophys Acta 2007;1768:1006–18. Sands WA, Palmer TM. Regulating gene transcription in response to cyclic AMP elevation. Cell Signal 2008;20:460–6. Schäfers RF, Adler S, Daul A, Zeitler G, Vogelsang M, Zerkowski HR, et al. Positive inotropic effects of the β2-adrenoceptor agonist terbutaline in the human heart: effects of long-term β1-adrenoceptor antagonist treatment. J Am Coll Cardiol 1994;23:1224–33. Shiojima I, Walsh K. Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway. Genes Dev 2006;20:3347–65. Siedlecka U, Arora M, Kolettis T, Soppa GK, Lee J, Stagg MA, et al. Effects of clenbuterol on contractility and Ca2+ homeostasis of isolated rat ventricular myocytes. Am J Physiol Heart Circ Physiol 2008;295:H1917–26. Soppa GK, Smolenski RT, Latif N, Yuen AH, Malik A, Karbowska J, et al. Effects of chronic administration of clenbuterol on function and metabolism of adult rat cardiac muscle. Am J Physiol Heart Circ Physiol 2005;288:H1468–76. Stanley WC, Recchia FA, Lopaschuk GD. Myocardial substrate metabolism in the normal and failing heart. Physiol Rev 2005;85:1093–129. The Xamoterol in Severe Heart Failure Study Group. Xamoterol in severe heart failure. Lancet 1990;336:1–6. Terpstra GK, Raaijmakers JA. Beta-agonistic properties of tulobuterol, a new betasympathicomimetic drug, and its effects on pulmonary beta-adrenoceptor characteristics. Lung 1990;168:179–85. Thomson DM, Herway ST, Fillmore N, Kim H, Brown JD, Barrow JR, et al. AMP-activated protein kinase phosphorylates transcription factors of the CREB family. J Appl Physiol 2008;104:429–38. van Bilsen M, van Nieuwenhoven FA, van der Vusse GJ. Metabolic remodelling of the failing heart: beneficial or detrimental? Cardiovasc Res 2009;81:420–8. Wu Z, Huang X, Feng Y, Handschin C, Feng Y, Gullicksen PS, et al. Transducer of regulated CREB-binding proteins (TORCs) induce PGC-1α transcription and mitochondrial biogenesis in muscle cells. Proc Natl Acad Sci U S A 2006;103:14379–84. Xiao RP, Ji X, Lakatta EG. Functional coupling of the β2-adrenoceptor to a pertussis toxin-sensitive G protein in cardiac myocytes. Mol Pharmacol 1995;47:322–9. Xiao RP, Zhu W, Zheng M, Chakir K, Bond R, Lakatta EG, et al. Subtype-specific βadrenoceptor signaling pathways in the heart and their potential clinical implications. Trends Pharmacol Sci 2004;25:358–65. Yamamoto DL, Hutchinson DS, Bengtsson T. β2-adrenergic activation increases glycogen synthesis in L6 skeletal muscle cells through a signaling pathway independent of cyclic AMP. Diabetologia 2007;50:158–67. Yoo B, Lemaire A, Mangmool S, Wolf MJ, Curcio A, Mao L, et al. β1-adrenergic receptors stimulate cardiac contractility and CaMKII activation in vivo and enhance cardiac dysfunction following myocardial infarction. Am J Physiol Heart Circ Physiol 2009;297:H1377–86. Zhu WZ, Zheng M, Koch WJ, Lefkowitz RJ, Kobilka BK, Xiao RP. Dual modulation of cell survival and cell death by β2-adrenergic signaling in adult mouse cardiac myocytes. Proc Natl Acad Sci U S A 2001;98:1607–12.