Phosphatidylinositol 3-Kinase Functionally Compartmentalizes the Concurrent Gs Signaling During β2-Adrenergic Stimulation
Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of β2-adrenoceptor (β2-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of β2-AR-coupled Gi proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the β2-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the β2-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables β2-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in β2-AR-induced cAMP formation. Blocking Gi or Gβγ signaling with pertussis toxin or βARK-ct, a peptide inhibitor of Gβγ, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of β2-AR-PKA signaling sequentially involves Gi, Gβγ, and PI3K. Thus, PI3K constitutes a key downstream event of β2-AR-Gi signaling, which confines and negates the concurrent β2-AR/Gs-mediated PKA signaling.
- cAMP signal compartmentation
- phosphatidylinositol 3-kinase
- cardiac contractility
Afundamental question of G protein-coupled receptor (GPCR) signal transduction is how a myriad of GPCRs and numerous cognate G proteins can elicit highly specific physiological responses. One of the most notable determinants of the specificity and effector-selectivity of receptor signaling is the cellular compartment over which the signal can transmit. As a prototypical GPCR, β-adrenoceptor (AR) activates the classical Gs-adenylyl cyclase (AC)-cAMP-protein kinase A (PKA) signaling cascade. In the heart, PKA subsequently phosphorylates a multitude of regulatory proteins involved in cardiac muscle contraction, including sarcolemmal L-type Ca2+ channel,1 the sarcoplasmic reticulum (SR) Ca2+ pump regulator phospholamban (PLB),2 and myofilament proteins,3 resulting in increased contractility and accelerated cardiac relaxation.
However, a large body of evidence indicates that β2-AR-induced cAMP/PKA signaling is tightly localized to the cell surface membrane microdomains in the vicinity of L-type Ca2+ channels and cannot transmit to non-sarcolemmal target proteins, whereas β1-AR-mediated cAMP/PKA signaling can broadcast throughout the cell.4 Specifically, β1-AR can activate sarcolemmal L-type Ca2+ channels and increase phosphorylation of multiple intracellular proteins, such as PLB at SR membrane and troponin I and C proteins of myofilaments, resulting in both positive contractile and relaxant responses.5–12⇓⇓⇓⇓⇓⇓⇓ In contrast, β2-AR stimulation selectively activates the Ca2+ channel, without affecting the aforementioned intracellular PKA target proteins, leading to a positive inotropic effect in the absence of a relaxant effect.4,7,13⇓⇓ Moreover, studies with patch-clamp single-channel recordings have shown that in both cardiac myocytes14 and hippocampal neurons,15 β2-AR stimulation modulates single L-type Ca2+ channel activity only in a local mode (agonist included within the patch pipette with tip diameter ≈1.0 μm) and not in a remote mode (agonist perfused outside the patch), whereas β1-AR stimulation increases the channel activity in both modes.
The spatial and functional restriction of β2-AR/Gs-induced cAMP/PKA signaling might be explained by the additional coupling of the receptor to Gi proteins.8,12,16⇓⇓ Inhibition of Gi signaling with pertussis toxin (PTX) allows β2-AR stimulation to induce PLB phosphorylation5 and to modulate single L-type Ca2+ channels in the remote mode.14 Although the Gi pathway is implicated in the functional compartmentation of β2-AR-mediated cAMP/PKA signaling, the downstream events of the β2-AR-Gi pathway remain largely unknown.
Phosphoinositide 3-kinases (PI3Ks) are group of enzymes exhibiting both lipid kinase and protein kinase activities, and are broadly engaged in multiple vital cellular functions, including cell proliferation, cytoskeletal organization, cell migration, cell survival and cell growth.17–21⇓⇓⇓⇓ Based on their structure and substrate specificity, PI3Ks can be divided into 3 classes (I, II, and III), and the class I PI3Ks can be further categorized into 2 subclasses (A and B). The class IB PI3K (also known as PI3Kγ) can be activated by Gβγ subunits of G proteins.22 Our recent studies have shown that PI3K constitutes a downstream mediator of β2-AR-Gi signaling to deliver a cell survival signal, which protects heart muscle cells against a wide range of apoptotic insults such as hypoxia, reactive oxygen species, and enhanced β1-AR stimulation.23,24⇓ However, to date, there is no information available regarding the possible involvement of PI3K in acute modulation of cardiac function. Moreover, it remains unexplored whether PI3K participates in the Gi-dependent functional compartmentation of β2-AR-PKA signaling, thereby reshaping the PKA-mediated protein phosphorylation and cardiac functional modulation.
The goal of the present investigation is to determine the possible involvement of PI3K in the functional restriction and effector-selectivity of the β2-AR/Gs-mediated PKA signaling. We show that inhibition of PI3K by PI3K inhibitors enables β2-AR-PKA signaling to reach intracellular proteins, as evidenced by a robust increase in PKA-dependent phosphorylation of PLB, a major regulator of cardiac relaxation, and that overexpression of constitutively active PI3K reduces basal phosphorylation of PLB. In the presence of the PI3K inhibitor, β2-AR-induced PLB phosphorylation is associated with a de novo relaxant effect and an increase in the receptor-mediated positive contractile response. These potentiating effects induced by PI3K inhibition are fully prevented by disrupting Gi or Gβγ signaling, suggesting that the pathway linking Gi activation to the functional compartmentation of β2-AR-activated PKA signaling sequentially involves Gi, Gβγ, and PI3K.
Materials and Methods
Measurements of Cell Length and Intracellular Ca2+ Transients
Single ventricular myocytes were isolated from 2- to 4-month-old rat hearts by a standard enzymatic technique.25 Animals were provided by Charles River Laboratories (Wilmington, Mass), and the protocol of the study was approved by the Animal Care and Use Committee of the Gerontology Research Center, National Institute on Aging. In some experiments, heart cells were treated with 1.5 μg/mL PTX (Sigma) for 3 hours at 37°C to block Gi signaling, as described previously.8 In another subset experiments, myocytes were loaded with a fluorescent Ca2+ probe, indo-1/acetoxymethyl-ester (Molecular Probes), and then electrically paced at 0.5 Hz at 23°C, as described previously.25
PI3K Activity Assay
Suspensions of adult rat cardiomyocytes were first incubated with appropriate pharmacological agents. Measurement of PI3K activity was then performed, as previously described.23
Measurements of cAMP Accumulation and PKA-Dependent Phosphorylation of Phospholamban at Ser16
Intracellular cAMP was measured, as previously described.28 PKA-dependent phosphorylation of phospholamban (PLB) at Ser16 was assayed as described previously.5,10⇓ Protein concentration was determined by the method of Lowry et al, 26 using ovalbumin as standard. After electrophoresis, the Western blot was performed as described previously.5,10⇓
Culture and Adenoviral Infection of Adult Rat Ventricular Myocytes
In some experiments, heart cells were cultured and infected with adenovirus-p110* (constitutively active PI3K),27 adenovirus-βARK-ct (an adenovirus vector encoding βARK1 carboxyl-terminal domain, which blocks Gβγ signaling, kindly provided by R.J. Lefkowitz and W.J. Koch at Duke University, Durham, NC) or adenovirus-β-gal (an adenovirus vector expressing lacZ, as a negative control), all at multiplicity of infection (moi) of 100, as previously described.24 All experiments were performed after 24 hours of adenoviral infection.
All quantitative data are expressed as mean±SE. The results were analyzed using ANOVA. Differences were considered significant when values were P<0.05.
β2-AR Stimulation Increases PI3K Activity
To determine the role of PI3K in the functional compartmentation of β2-AR signaling, we first examined the possible effect of β2-AR stimulation on PI3K activity using a lipid kinase assay to monitor the conversion of PI into PI-P.23 As shown in Figure 1, β2-AR stimulation by zinterol for 15 minutes increases PI-P production by ≈2.5-fold, and the addition of a highly selective β1-AR antagonist, CGP20712A (0.3 μmol/L), does not alter this effect (data not shown). Stimulation of insulin-like growth factor-1 for 15 minutes, a known PI3K activator,29 is used as a positive control. LY294002 (5 μmol/L for 15 minutes), a PI3K inhibitor,30 completely blocks agonist-induced increase in PI3K activity. This result is consistent with our previous findings that β2-AR stimulation activates PI3K in mouse and neonatal rat cardiac myocytes.23,24⇓
Inhibition of PI3K Enhances β2-AR-Mediated Contractile Response
Next, we evaluated the potential involvement of PI3K in β2-AR-mediated cardiac functional modulation with respect to single cell contraction and intracellular Ca2+ (Cai) handling. The PI3K inhibitor, LY294002, whereas it effectively blocks agonist-induced PI3K activation (Figure 1), has no significant effect on the amplitude and kinetics of baseline myocyte contraction (Table). However, LY294002 markedly enhances β2-AR-mediated increase in cell contractility. Figure 2A shows a typical example of the β2-AR agonist zinterol-elicited increase in contraction amplitude in the absence and presence of the PI3K inhibitor. The potentiating effect of LY294002 on β2-AR contractile response occurs rapidly and is fully reversible on washout. Figure 2B illustrates the average dose-response of zinterol. The PI3K inhibitor shifts the whole dose-response curve upward and leftward, thus reducing the EC50 of zinterol from 3×10−7 to 5×10−8 mol/L.
To further determine the possible effect of PI3K inhibition on β2-AR-induced change in Cai transient, indexed by indo-1 fluorescent ratio,25 we simultaneously measured the Cai transient and contraction in freshly isolated adult rat cardiomyocytes loaded with indo-1 (Figure 2C). In the absence of LY294002, β2-AR stimulation by zinterol (0.1 μmol/L, a concentration of ≈EC50) increases the amplitudes of contraction and Cai transient to 162.3±13.2% and 124.4±4.0% of control, respectively (Figures 2C and 2D). Although LY294002 alone has no significant effect on either parameter (Table 1), it enhances zinterol-induced increases in the contraction and Cai transient by ≈2-fold and ≈1.5-fold, respectively (Figures 2C and 2D, also see Figures 7A and 2⇑B). This result indicates that PI3K, known as a key molecule mediating the β2-AR antiapoptotic effect,23,24⇓ can acutely regulate cardiac contraction and Cai handling during β2-AR stimulation.
Inhibition of PI3K Enables β2-AR Stimulation to Induce a De Novo Relaxant Effect
In addition to augmenting β2-AR-induced positive inotropic effect, inhibition of PI3K by LY294002 overtly augments the effects of β2-AR on the kinetics of contraction and Cai transient. As shown in Figure 2E, zinterol alone only slightly reduces the half relaxation time (t1/2) of contraction and has virtually no effect on the t1/2 of Cai transient, consistent with previous reports.5–8,13⇓⇓⇓⇓ However, in the presence of LY294002, β2-AR stimulation causes a robust relaxant effect, as manifested by the significantly abbreviated t1/2 of both parameters (84.7±1.3% and 84.5±4.1% of control for contraction and Cai transient, respectively) (Figures 2C and 2E; also see Figures 7C and 7D). Because cardiac relaxant effect is a hallmark of PKA-dependent phosphorylation of PLB, the present data suggests that inhibition of PI3K permits β2-AR stimulation to evoke a de novo relaxant effect, likely by affecting phosphorylation status of PLB, the major regulator of cardiac SR Ca2+ pump.2 To test this hypothesis, we next measured β2-AR-induced PLB phosphorylation in the presence or absence of PI3K inhibition.
PI3K Inhibition Enables β2-AR Stimulation to Increase Phosphorylation of PLB at Ser16
Previous studies have shown that PLB is phosphorylated by β1-AR stimulation at 2 adjacent sites, Ser16 and Thr17, catalyzed by PKA and Ca2+/calmodulin-dependent kinase II, respectively,2,31⇓ whereas β2-AR stimulation is unable to induce phosphorylation of PLB and other cytosolic regulatory proteins.5,7,9,10⇓⇓⇓ Consistent with the previous notion, the β2-AR agonist, zinterol, has only a very minor effect on PKA-dependent phosphorylation of PLB at Ser16. A typical Western blot and the average dose-response relationship of β2-AR-induced phosphorylation of PLB at Ser16 are shown in Figures 3A and 3B, respectively. Zinterol even at a maximal concentration (10 μmol/L) only increases Ser16-PLB phosphorylation by 1.2-fold. In sharp contrast, in LY294002-pretreated myocytes, β2-AR stimulation by zinterol results in a dose-dependent increase in PLB phosphorylation, with a maximal effect of 3.8-fold increase and an EC50 of 3.2×10−7 mol/L. The inhibitory effect of PI3K on β2-AR-induced PLB phosphorylation is further confirmed by using another PI3K inhibitor, wortmannin.32 Similar to LY294002, wortmannin also allows the β2-AR agonist, zinterol, to increase PKA-dependent phosphorylation of PLB at Ser16 in a dose-dependent manner (Figures 3A and 3B). These results indicate that inhibition of PI3K enables β2-AR to induce PLB phosphorylation at Ser16, thus resulting in a de novo relaxant effect. This provides the first evidence that PI3K is critically involved in the functional compartmentation and effector-selectivity of β2-AR signaling.
Overexpression of Constitutively Active PI3K Decreases Phosphorylation of PLB at Ser16
To further examine the possible role of PI3K on PKA-dependent PLB phosphorylation, we expressed a constitutively active PI3K (Adv-p110*) in cultured adult rat cardiomyocytes using adenoviral gene transfer. In myocytes overexpressing the constitutively active PI3K, basal phosphorylation of PLB at Ser16 is reduced by ≈50% as compared with that in myocytes-infected by a control virus (Adv-β-gal, at the same moi) (Figures 4A and 4B), suggesting that activation of PI3K per se inhibits PKA-dependent phosphorylation of PLB. Interestingly, in myocytes expressing the constitutively active PI3K, pretreatment of cells with LY294002 (5 μmol/L for 10 minutes) cannot unmask β2-AR-induced PLB phosphorylation, whereas it fully abolishes agonist-induced endogenous PI3K activation (Figure 1). This is consistent with the fact that LY294002 (5 μmol/L) is unable to inhibit the constitutively active PI3K-induced phosphorylation of Akt, a downstream effect of PI3K (data not shown, also see Wu et al27). This result indicates that the potentiating effect of LY294002 on β2-AR-induced PLB phosphorylation at Ser16 is mediated specifically by inhibiting of PI3K activity.
Inhibition of PI3K by LY294002 Cannot Enhance β2-AR-Induced cAMP Formation
To understand the mechanism underlying the potentiating effects of PI3K inhibition on β2-AR-induced PLB phosphorylation, we next measured intracellular cAMP formation in response to β2-AR stimulation in the presence and absence of the PI3K inhibitor, LY294002 (Figure 5). β2-AR stimulation by zinterol (0.1 μmol/L, a concentration of ≈EC50) significantly increases cAMP accumulation by ≈2-fold. However, pretreatment of cells with LY294002 cannot further enhance zinterol-induced increase in intracellular cAMP accumulation. This result suggests that the confining effect of β2-AR-PI3K on the concurrent β2-AR-Gs signaling is likely mediated by activating protein phosphatases, rather than by inhibiting formation of cAMP.
Disruption of Gi Signaling and Inhibition of PI3K Have Nonadditive Effect on β2-AR-Induced Phosphorylation of PLB at Ser16
We hypothesized that if PI3K activation is the downstream event of Gi signaling, disruption of Gi signaling by PTX treatment and inhibition of PI3K should have nonadditive effect on β2-AR-induced phosphorylation of PLB at Ser16. Indeed, pretreatment of cells with both PTX and LY294002 exhibits no additive effect on PLB phosphorylation in response to the β2-AR agonist, zinterol (0.1 μmol/L, a dose the ≈EC50 used to avoid saturation). Cotreatment of cells with PTX and LY294002 enhances the response of PLB phosphorylation by ≈2.2-fold, similar to the single treatment of cells by either PTX or LY294002 (Figures 6A and 6B). The same conclusion is drawn when even a lower dose of zinterol (10 nmol/L) is utilized (1.8±0.4-, 1.9±0.4-, and 1.8±0.5-fold increase for PTX+zinterol, LY294002+zinterol, and PTX+LY294002+zinterol, respectively, n=6, P>0.05 among groups), indicating that the nonadditive effect is not due to a saturation of PLB phosphorylation. Taken together, these results strongly suggest that the additional Gi coupling offsets β2-AR-stimulated PLB phosphorylation via a PI3K-dependent mechanism.
Disruption of Gi Signaling Prevents the Potentiating Effects Induced by PI3K Inhibition
In PTX-treated cells, the PI3K inhibitor fails to further enhance the β2-AR-induced augmentation in contraction amplitude (Figure 7A), consistent with the data on PLB phosphorylation described above. This is not caused by a saturation of cell contractile response, because the contraction amplitude can be increased by about 4-fold in response to a full β-AR agonist, isoproterenol (1 μmol/L) (data not shown). Likewise, LY294002 does not significantly alter the response of Cai transient in those PTX-treated cells (Figure 7B). Furthermore, PTX treatment prevents LY294002 to potentiate the β2-AR-mediated abbreviation in the t1/2 of contraction or Cai transient (Figures 7C and 7D). Thus, inhibition of Gi signaling completely abolishes the potentiating effects of the PI3K inhibitor on β2-AR-mediated positive contractile and relaxant responses. This indicates that β2-AR stimulation activates PI3K signaling via a Gi-dependent mechanism.
β2-AR-Mediated PI3K Activation Requires Gβγ Signaling
We next sought to discriminate which subunit of heterotrimeric Gi proteins is essential to the PI3K activation by infecting myocytes with either adenovirus-βARK-ct (βARK-ct, a peptide inhibitor of Gβγ signaling)33 or adenovirus-β-gal (as a negative control). It is noteworthy that the basal contraction or Cai transient in cells infected with adenovirus-βARK-ct is comparable to that in myocytes infected by the control virus, adenovirus-β-gal (Table). Figure 7E shows that in adenovirus-β-gal-infected myocytes inhibition of PI3K by LY294002 augments zinterol-induced increase in the contraction amplitude, similar to the situation in freshly isolated myocytes (Figures 2 and 7⇑A). However, in myocytes infected by adenovirus-βARK-ct, LY294002 fails to further enhance β2-AR-evoked positive inotropic effect. Similarly, inhibition of Gβγ signaling prevents LY294002 from potentiating the β2-AR-mediated increase in the Cai transient (Figure 7F). These observations suggest that Gβγ subunits dissociated from Gi proteins are critically involved in β2-AR-mediated activation of PI3K in cardiac myocytes.
PI3K Functionally Restricts β2-AR-Mediated cAMP/PKA Signaling
Our present study shows that inhibition of PI3K also enables β2-AR to induce PLB phosphorylation as well as a de novo relaxant response in adult rat cardiomyocytes, and that overexpression of a constitutively active PI3K significantly decreases the basal phosphorylation of PLB. The potentiating effects caused by PI3K inhibition are completely prevented by blocking either Gi or Gβγ signaling, indicating that the Gi-dependent restriction of β2-AR-PKA signaling involves a Gi-Gβγ-PI3K pathway. Thus, in addition to its essential roles in the regulation of chronic cellular processes such as proliferation, cell migration, cell survival, and cell growth, PI3K constitutes an important molecular link responsible for the functional compartmentation of β2-AR/Gs-PKA signaling, negating the β2-AR/Gs-mediated phosphorylation of PKA target proteins and the positive cardiac contractile and relaxant responses.
Possible Mechanisms Underlying the Inhibitory Effect of PI3K on β2-AR-PKA Signaling
The present result demonstrates that the PI3K inhibitor LY294002 markedly enhances the response of contraction or Cai transient to β2-AR stimulation (Figure 2). This enhancement can be largely explained by the increase in PLB phosphorylation at Ser16 (Figure 3). It is widely accepted that increased PKA-dependent phosphorylation of PLB not only accelerates SR Ca2+ uptake, but also elevates SR Ca2+ content due to the increased Ca2+ pump activity,2 thereby leading to hastened cardiac relaxation and increased cardiac contractility. This is supported by the notion that in a PLB-deficient mouse model (a functional equivalent of PLB full phosphorylation), both cardiac contractility and relaxation velocity are significantly enhanced as compared with that in wild-type animals.34 Alternatively, inhibition of PI3K might enhance β2-AR-induced increase in L-type Ca2+ currents, which could elevate the amplitudes of Ca2+ transient and contraction. However, it has been recently shown that activation of PI3K increases the L-type Ca2+ current in vascular smooth muscle cells.35 Nevertheless, the possible effect of PI3K on cardiac Ca2+ currents awaits future investigation.
It has been demonstrated that PI3K exerts both lipid kinase and protein kinase activities.17,18⇓ Interestingly, the present results show that inhibition of PI3K markedly enhances β2-AR-induced phosphorylation of PLB at Ser16 by PKA (Figure 3), suggesting that PI3K may cause dephosphorylation of phosphorylated PLB-Ser16. Because inhibition of PI3K by LY294002 does not affect zinterol-induced increase in cAMP formation (Figure 5) and inhibition of Gi signaling does not attenuate β2-AR/Gs-induced increase in total cellular cAMP production13 or in PKA activity,5 the inhibitory effect of PI3K on β2-AR-evoked PLB phosphorylation might occur downstream of cAMP/PKA, perhaps via direct or indirect activation of certain protein phosphatases. In this regard, emerging evidence suggests that β2-AR is able to form a macromolecular signaling complex with protein phosphatase 2A.15 Moreover, calyculin A, an inhibitor of protein phosphatase 1 and 2A, potentiates β2-AR-induced positive inotropic effect in a PTX-sensitive manner,5 suggesting that protein phosphatases might be engaged in β2-AR/Gi signaling. In this scenario, PI3K might act as a protein kinase to increase protein phosphatase activity directly via phosphorylation or indirectly through phosphorylation and subsequent inactivation of endogenous protein phosphatase inhibitor-I. Further study is merited to address these issues.
Regarding the involvements of specific PI3K isoenzymes in β2-AR/Gi signaling, previous studies have shown that the class IB PI3K (PI3Kγ isoform) is a downstream target of Gβγ signaling.22 However, recent studies suggest that in cultured neonatal rat cardiac myocytes, stimulation of β-AR (subtype not specified) may activate PI3Kα and β.36 Because of the lack of isoform-specific PI3K inhibitors, the present study cannot discriminate the specific isoenzymes of PI3K family involved in β2-AR-coupled Gi signaling in adult rat cardiac myocytes.
Potential Involvement of PI3K in Agonist-Induced β2-AR Desensitization and Its Pathophysiological Relevance
As is true for all of GPCRs, the exposure of a receptor to an agonist results in rapid diminishment in its signaling efficiency (referred to as desensitization). Agonist-dependent β2-AR desensitization is initiated by phosphorylation of the activated receptor by members of the GPCR kinase family, particularly the β-adrenergic receptor kinase-1 (βARK1).37 It is noteworthy that PI3K is able to physically associate with βARK1 and promote agonist-dependent β-AR internalization,38 perhaps contributing to agonist-dependent β-AR desensitization. The robust inhibitory effect of PI3K activation on β2-AR-induced positive contractile and relaxant responses, demonstrated by the present results, raises an intriguing and important question regarding the possible requirement of PI3K for agonist-induced β2-AR desensitization.
Although activation of β2-AR/Gi-mediated PI3K pathway protects cardiac myocytes against apoptosis,23,24⇓ an imbalance of β2-AR-initiated Gs and Gi signaling cascades may have pathological consequences. Both recent in vivo and in vitro studies have demonstrated that activation of PI3K signaling is coincident with heart muscle cell hypertrophy in response to pressure overload or β-AR stimulation.36,39⇓ In addition, in many kinds of chronic heart failure in human and animal models, the contractile response to β-AR stimulation is markedly diminished,40,41⇓ which is accompanied by enhanced Gi signaling.42 It is speculated that the upregulation of β2-AR-Gi signaling in the functionally compensated hypertrophic heart or in the early stages of heart failure may protect against myocyte apoptosis and consequently slow the progression of cardiomyopathy and contractile dysfunction. However, the exaggerated β2AR-Gi signaling may blunt the Gs-mediated contractile support, contributing to the phenotype of decompensated heart failure.
This work is supported by NIH intramural research grant (R.-P.X.). The authors would like to thank Drs H. Cheng and E.G. Lakatta for stimulating discussions, and Dr H. Spurgeon and B. Ziman for their excellent technique support.
Original received February 1, 2002; revision received May 20, 2002; accepted May 20, 2002.
- ↵Koss KL, Kranias EG. Phospholamban. a prominent regulator of myocardial contractility. Circ Res. 1996; 79: 1059–1063.
- ↵Garvey JL, Kranias EG, Solaro RJ. Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts. Biochem J. 1988; 249: 709–714.
- ↵Xiao RP. β-Adrenergic signaling in the heart: dual coupling of the β2-adrenergic receptor to Gs and Gi proteins. Sci STKE. 2001; 104: 16.
- ↵Kuschel M, Zhou YY, Cheng H, Zhang SJ, Chen Y, Lakatta EG, Xiao RP. Gi protein-mediated functional compartmentalization of cardiac β2-adrenergic signaling. J Biol Chem. 1999; 274: 22048–22052.
- ↵Xiao RP, Lakatta EG. β1-Adrenoceptor stimulation and β2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2=, and Ca2= current in single rat ventricular cells. Circ Res. 1993; 73: 286–300.
- ↵Xiao RP, Hohl C, Altschuld R, Jones L, Livingston B, Ziman B, Tantini B, Lakatta EG. β2-Adrenergic receptor-stimulated increase in cAMP in rat heart cells is not coupled to changes in Ca2= dynamics, contractility, or phospholamban phosphorylation. J Biol Chem. 1994; 269: 19151–19156.
- ↵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: 322–329.
- ↵Altschuld RA, Starling RC, Hamlin RL, Billman GE, Hensley J, Castillo L, Fertel RH, Hohl CM, Robitaille PM, Jones LR, Xiao RP, Lakatta EG. Response of failing canine and human heart cells to β2-adrenergic stimulation. Circulation. 1995; 92: 1612–1618.
- ↵Kuschel M, Zhou YY, Spurgeon HA, Bartel S, Karczewski PZS, Krause EG, Lakatta EG, Xiao RP. β2-adrenergic cAMP signaling is uncoupled from phosphorylation of cytoplasmic proteins in canine heart. Circulation. 1999; 99: 2458–2465.
- ↵Xiao RP, Cheng H, Zhou YY, Kuschel M, Lakatta EG. Recent advances in cardiac β2-adrenergic signal transduction. Circ Res. 1999; 85: 1092–1100.
- ↵Xiao RP, Avdonin P, Zhou YY, Cheng H, Akhter SA, Eschenhagen T, Lefkowitz RJ, Koch WJ, Lakatta EG. Coupling of β2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes. Circ Res. 1999; 84: 43–52.
- ↵Davare MA, Avdonin V, Hall DD, Peden EM, Burette A, Weinberg RJ, Horne M C, Hoshi T, Hell JW. A β2-adrenergic receptor signaling complex assembled with the Ca2= channel Cav1.2. Science. 2001; 293: 98–101.
- ↵Bondeva T, Pirola L, Bulgarelli-Leva G, Rubio I, Wetzker R, Wymann MP. Bifurcation of lipid and protein kinase signals of PI3Kγ to the protein kinases PKB and MAPK. Science. 1998; 282: 293–296.
- ↵Ma AD, Metjian A, Bagrodia S, Taylor S, Abrams CS. Cytoskeletal reorganization by G protein-coupled receptors is dependent on phosphoinositide 3-kinase γ, a Rac guanosine exchange factor, and Rac. Mol Cell Biol. 1998; 18: 4744–4751.
- ↵Servant G, Weiner OD, Herzmark P, Balla T, Sedat JW, Bourne HR. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science. 2000; 287: 1037–1040.
- ↵Chesley A, Lundberg MS, Asai T, Xiao RP, Ohtani S, Lakatta EG, Crow MT. The β2-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through Gi-dependent coupling to phosphatidylinositol 3′-kinase. Circ Res. 2000; 87: 1172–1179.
- ↵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: 1607–1612.
- ↵Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem. 1951; 193: 265–275.
- ↵Wu W, Lee WL, Wu YY, Chen D, Liu TJ, Jang A, Sharma PM, Wang PH. Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase 3 and apoptosis of cardiac muscle cells. J Biol Chem. 2000; 275: 40113–40119.
- ↵Zhou YY, Yang D, Zhu WZ, Zhang SJ, Wang DJ, Rohrer DK, Devic E, Kobilka BK, Lakatta EG, Cheng H, Xiao RP. Spontaneous activation of β2- but not β1-adrenoceptors expressed in cardiac myocytes from β1β2 double knockout mice. Mol Pharmacol. 2000; 58: 887–894.
- ↵Duan C, Bauchat JR, Hsieh T. Phosphatidylinositol 3-kinase is required for insulin-like growth factor-I-induced vascular smooth muscle cell proliferation and migration. Circ Res. 2000; 86: 15–23.
- ↵Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994; 269: 5241–5248.
- ↵Arcaro A, Wymann MP. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J. 1993; 296: 297–301.
- ↵Koch WJ, Hawes BE, Allen LF, Lefkowitz RJ. Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by Gβγ activation of p21ras. Proc Natl Acad Sci U S A. 1994; 91: 12706–12710.
- ↵Chu G, Luo W, Slack JP, Tilgmann C, Sweet WE, Spindler M, Saupe KW, Boivin GP, Moravec CS, Matlib MA, Grupp IL, Ingwall JS, Kranias EG. Compensatory mechanisms associated with the hyperdynamic function of phospholamban-deficient mouse hearts. Circ Res. 1996; 79: 1064–1076.
- ↵Macrez N, Mironneau C, Carricaburu V, Quignard JF, Babich A, Czupalla C, Nurnberg B, Mironneau J. Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca2= channels. Circ Res. 2001; 89: 692–699.
- ↵Morisco C, Zebrowski D, Condorelli G, Tsichlis P, Vatner SF, Sadoshima J. The Akt-glycogen synthase kinase 3β pathway regulates transcription of atrial natriuretic factor induced by β-adrenergic receptor stimulation in cardiac myocytes. J Biol Chem. 2000; 275: 14466–14475.
- ↵Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, Lefkowitz RJ. Cardiac function in mice overexpressing the β-adrenergic receptor kinase or a βARK inhibitor. Science. 1995; 268: 1350–1353.
- ↵Naga Prasad SV, Barak LS, Rapacciuolo A, Caron MG, Rockman HA. Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by β-adrenergic receptor kinase 1: a role in receptor sequestration. J Biol Chem. 2001; 276: 18953–18959.
- ↵Naga Prasad SV, Esposito G, Mao L, Koch WJ, Rockman HA. Gβγ-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy. J Biol Chem. 2000; 275: 4693–4698.
- ↵Bristow MR, Ginsburg R, Umans V, Fowler M, Minobe W, Rasmussen R, Zera P, Menlove R, Shah P, Jamieson S. β1- and β2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective β1-receptor down-regulation in heart failure. Circ Res. 1986; 59: 297–309.
- ↵Eschenhagen T, Mende U, Nose M, Schmitz W, Scholz H, Haverich A, Hirt S, Doring V, Kalmar P, Hoppner W. Increased messenger RNA level of the inhibitory G protein α subunit Giα-2 in human end-stage heart failure. Circ Res. 1992; 70: 688–696.