Published online before print November 11, 2004, doi: 10.1161/01.RES.0000150049.74539.8a
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© 2004 American Heart Association, Inc.
Cellular Biology |
Phosphatidylinositol 3-Kinase Offsets cAMP-Mediated Positive Inotropic Effect via Inhibiting Ca2+ Influx in Cardiomyocytes
From the Laboratory of Cardiovascular Science (V.L., S.-H.J., K.C., V.M., M.Z., M.T.C., W.W., E.G.L., R.-P.X.), Gerontology Research Center, National Institute on Aging, NIH, Baltimore, Md; The Institute of Molecular Medicine and The Institute of Cardiovascular Sciences (M.Z., R.-P.X.), Peking University, Beijing, China. Present address for V.L. is Laboratoire de Pharmacologie de l’UFR de Pharmacie & INSERM EMI0356, Université Victor Segalen Bordeaux 2, Bordeaux, France; and for S.-H.J., Department of Physiology, Cheju National University College of Medicine, Ara 1-Dong, Jeju, Korea.
Correspondence to Rui-Ping Xiao, MD, PhD, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, 5600 Nathan Shock Dr, Baltimore, MD 21224. E-mail XiaoR{at}grc.nia.nih.gov
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Abstract |
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Phosphoinositide 3-kinase (PI3K) has been implicated in ß2-adrenergic receptor (ß2-AR)/Gi-mediated compartmentation of the concurrent Gs-cAMP signaling, negating ß2-AR–induced phospholamban phosphorylation and the positive inotropic and lusitropic responses in cardiomyocytes. However, it is unclear whether PI3K crosstalks with the ß1-AR signal transduction, and even more generally, with the cAMP/PKA pathway. In this study, we show that selective ß1-AR stimulation markedly increases PI3K activity in adult rat cardiomyocytes. Inhibition of PI3K by LY294002 significantly enhances ß1-AR–induced increases in L-type Ca2+ currents, intracellular Ca2+ transients, and myocyte contractility, without altering the receptor-mediated phosphorylation of phospholamban. The LY294002 potentiating effects are completely prevented by ßARK-ct, a peptide inhibitor of ß-adrenergic receptor kinase-1 (ßARK1) as well as Gß
signaling, but not by disrupting Gi function with pertussis toxin. Moreover, forskolin, an adenylyl cyclase activator, also elevates PI3K activity and inhibition of PI3K enhances forskolin-induced contractile response in a ßARK-ct sensitive manner. In contrast, PI3K inhibition affects neither the basal contractility nor high extracellular Ca2+-induced increase in myocyte contraction. These results suggest that ß1-AR stimulation activates PI3K via a PKA-dependent mechanism, and that Gß and the subsequent activation of ßARK1 are critically involved in the PKA-induced PI3K signaling which, in turn, negates cAMP-induced positive inotropic effect via inhibiting sarcolemmal Ca2+ influx and the subsequent increase in intracellular Ca2+ transients, without altering the receptor-mediated phospholamban phosphorylation, in intact cardiomyocytes.
Key Words: PI3K • PKA • cardiac contractility • L-type calcium current • ß1-adrenergic receptor
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phosphoinositide 3-kinase (PI3K) family has been implicated in multiple vital cellular functions, including cell survival, cell proliferation, cytoskeletal remodeling, and vesicle trafficking.1, 2 Based on their structure and substrate specificity, PI3Ks are divided into three classes (I, II, and III). The most well characterized class I PI3Ks can be further categorized into two subgroups, IA and IB. Activation of class IA PI3Ks is controlled by stimulation of various receptors with intrinsic or associated tyrosine kinase activity, and class IB (also known as PI3K
) by activation of G protein–coupled receptors (GPCRs) via Gß subunits, although some exceptions have been reported.3,4
Increasing evidence suggests that PI3Ks are involved in cardiac ß2-adrenergic receptor (ß2-AR) signaling. In rodent cardiac myocytes, ß2-AR stimulation delivers an antiapoptotic signal through ß2-AR–activated Gi-Gß-PI3K-Akt pathway.5,6 More recent studies have demonstrated that PI3K also constitutes an important downstream signaling event of ß2-AR–coupled Gi to compartmentalize the ß2-AR/Gs-adenylyl cyclase (AC)-cAMP-PKA signaling, offsetting ß2-AR/Gs-mediated phosphorylation of phospholamban (PLB) and the positive inotropic and lusitropic effects in cardiomyocytes.7 However, it remains unknown whether PI3Ks are engaged in the signaling of ß1-AR, the predominant cardiac ß-AR subtype, and perhaps even more generalized signal transduction, eg, cAMP/PKA pathway, involved in the modulation of cardiac contractility. The main purpose of this study is to determine the possible involvement of PI3Ks in ß1-AR or PKA-mediated regulation of cardiac excitation-contraction coupling and the underlying mechanism.
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Materials and Methods |
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Cell Contraction, Intracellular Ca2+ Transient, and ICa Measurements
Ventricular cardiac myocytes were isolated from 2- to 4-month-old Sprague-Dawley rats, using a standard enzymatic technique.8 Cell contraction was indexed by the percent shortening of cell length after electrical stimulation at 0.5 Hz at 23°C, as previously described.8 In some experiments, cells were treated with 1.5 µg/mL PTX (Sigma Chemical Co) for 3 hours at 37°C.9 In another set of experiments, myocytes were loaded with a fluorescent Ca2+ probe, indo1-acetoxymethyl ester (Molecular Probes), to measure intracellular Ca2+ (Cai) transient, as described previously.8
ICa was measured via the whole-cell patch clamp technique using an Axopatch 1D amplifier (Axon Instruments Inc) in freshly isolated rat ventricular myocytes, as previously described.9 Briefly, cells were voltage-clamped at –40 mV to inactivate the sodium and T-type calcium channels. Potassium currents were eliminated by using K+ free solutions with tetraethylammonium (TEA). The patch pipette (1.4
1.8 M
) was filled with pipette solution containing (in mmol/L) CsCl 133, HEPES 5, TEA-Cl 20, MgATP 5, and EGTA 10 (pH 7.3, adjusted with CsOH), whereas myocytes were perfused with bath solution containing (in mmol/L) NaCl 140, CaCl2 1.8, CsCl 5.4, MgCl2 2, and HEPES 5 (pH 7.3, adjusted with NaOH).
Culture and Adenoviral Infection of Adult Rat Ventricular Myocytes
Myocytes were cultured and infected with adenovirus-ßARK-ct (an adenovirus vector carrying a gene encoding ß-AR kinase1 carboxyl-terminal fragment) or adenovirus-ß-gal (an adenovirus vector with a reporter gene lacZ, as a negative control), both at multiplicity of infection of 100. All experiments were performed after 24 hours of adenoviral infection.
PI3K Activity Assay
Suspensions of cardiac myocytes were first incubated with appropriate pharmacological agents. Measurement of PI3K activity was then performed, as previously described.5,10
PKA-Dependent Phosphorylation of Phospholamban at Ser16
PKA-dependent phosphorylation of PLB at Ser16 was assayed as described previously.11,12
Materials
Isoproterenol (ISO), norepinephrine (NE), prazosin, forskolin (FSK), insulin-like growth factor-1 (IGF-1), and ICI 118,551 (ICI) were purchased from Sigma Chemical Co and LY294002 from Calbiochem.
Statistical Evaluations
All data are presented as mean±SE of n number of experiments. Unpaired or paired Student t test, or ANOVA were used for statistical comparisons when appropriate, and differences were considered significant at P<0.05.
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Results |
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ß1-AR Stimulation Increases PI3K Activity in Freshly Isolated Adult Rat Ventricular Myocytes
We first examined the effect of ß1-AR stimulation on PI3K activity in adult rat ventricular myocytes, using a lipid kinase assay. As expected, IGF-1 stimulation, a known PI3K activator,13,14 elevated PI3K activity in a LY294002 (a PI3K inhibitor)-sensitive manner (Figure 1). Selective ß1-AR stimulation by NE (5x10–7 mol/L), in the presence of an
1-AR antagonist prazosin (10–5 mol/L) and a ß2-AR antagonist ICI 118,551 (10–7 mol/L), also markedly increased PI3K activity, similar to nonselective ß-AR stimulation with ISO (10–6 mol/L) (Figure 1). Interestingly, FSK (10–6 mol/L), a AC activator, also potently augmented PI3K activity to an extent similar to that induced by ß1-AR activation (Figure 1). These results indicate that activation of PKA or ß1-AR stimulation augments PI3K activity in intact cardiomyocytes.
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PI3K Signaling Negatively Regulates ß1-AR–Mediated Positive Contractile Response
We next determined whether activation of PI3Ks modulates ß-AR–mediated contractile response. Although inhibition of PI3K by LY294002 (5.10–6 mol/L) had only a minor effect on baseline contraction (107.8±1.4% of control, n=50 cells from 10 hearts; also see Figures 2 and 3), it clearly enhanced the contractile response to the nonselective ß-AR agonist, ISO, at a submaximal concentration (10–9 mol/L) (Figure 2A and 2B). The potentiating effect of LY294002 occurred regardless of the experimental protocol. When LY294002 was applied after ISO-mediated contractile response was stabilized, it further increased contraction amplitude from 164.90±12.42% to 208.02±16.63% of baseline (n=8, P<0.001; Figure 2C). The effect of LY294002 appeared rapidly and was completely reversible on washout. Likewise, in cells pretreated with LY294002 for 10 minutes, ISO increased the contraction amplitude to 188.87±18.77% of baseline (n=8, P<0.001; Figure 2D), which was comparable to the response induced by ISO plus LY294002 (Figure 2C). Another PI3K inhibitor, wortmannin also similarly enhanced ISO-mediated contractile response in adult rat cardiomyocytes (data not shown).
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P<0.001 as indicated. Baseline contraction amplitude is 6.05±0.42% of rest cell length (n=16 cells).
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Because stimulation of ß1-AR serves as the most predominant mechanism in regulating cardiac contractility in response to stress or exercise, we next measured the contractile response to selective ß1-AR stimulation by NE (5x10–8 mol/L, a concentration close to the EC50, plus prazosin at 10–5 mol/L and ICI 118,551 at 10–7 mol/L), in the presence or absence of the PI3K inhibitor. LY294002 administrated after ß1-AR stimulation further enhanced NE-induced contractile response (Figure 3A). Similar to the situation of mixed ß-AR stimulation with ISO, pretreatment of cells with LY294002 also significantly augmented NE-induced positive inotropic effect (Figure 3B). In both cases, the potentiating effects of LY294002 were completely reversible on washout (Figure 3A and 3B). As illustrated in Figure 3C, the whole concentration-response course of NE to increase cell contraction amplitude was markedly shifted leftward by LY294002 treatment. These results indicate that ß1-AR–mediated positive contractile response is negatively regulated by the receptor-activated PI3K signaling.
Surprisingly, the PI3K inhibitor augmented ß1-AR–positive inotropic effect without enhancing ß1-AR–mediated relaxant effect. In the absence and presence of LY294002, NE-induced abbreviation of 50% and 90% relaxation times (T50 and T90, respectively) were not significantly different (T50: 82.8±3.3% versus 84.7±6.1% of baseline; T90: 75.7±4.2% versus 78.8±4.7%). The inability of LY294002 to enhance NE-induced lusitropic effect is not attributable to the ß1-AR lusitropic effect already reaching the maximum, because NE at 10–6 mol/L caused a significantly greater abbreviation in T50 (74.1±6.5% versus 84.7±6.1%; n=5 to 8 cells; P<0.05).
Negative Inotropic Effect of PI3K Requires Activation of the cAMP/PKA Signaling
To define the potential target of PI3K in the ß1-AR signaling cascade, we evaluated the effect of PI3K inhibition on the contractile response to postreceptor manipulations: activation of AC by FSK (5x10–7 mol/L) or increasing intracellular Ca2+ by elevating extracellular Ca2+ concentration from 1.0 to 1.5 mmol/L (high extracellular [Ca2+]). Although both treatments increased the contraction amplitude to a comparable extent, LY294002 selectively enhanced the FSK-induced response, without affecting the high extracellular [Ca2+]-induced increase in myocyte contractility (Figure 4A and 4B). LY294002 also exhibited a moderate, but significant, potentiating effect on the contractile response to CPT-cAMP, an active cAMP analogue (data not shown). These results indicate that modulation of cardiomyocyte contractility by PI3K signaling requires the activation of the cAMP/PKA pathway. In addition, as is the case for ß1-AR stimulation, inhibition of PI3K augmented FSK-induced positive inotropic effect, but not its lusitropic effect (data not shown).
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PI3K Inhibition Does Not Affect ß1-AR Induced Phosphorylation of Phospholamban at Ser16
PLB, a primary regulator of the sarcoplasmic reticulum Ca2+ pump, can be phosphorylated at two adjacent sites, Ser16 and Thr17, by PKA and Ca2+/calmodulin-dependent kinase II, respectively, resulting in positive inotropic and lusitropic effects.15,16 We found no detectable effect of the PI3K inhibitor, LY294002, on ß1-AR–induced phosphorylation of PLB at Ser16 (Figure 5A and 5B). The concentration-response curves of NE-induced increase in PLB phosphorylation in the absence and presence of the PI3K inhibitor virtually overlapped with each other. This result is consistent with the inability of LY294002 to augment ß1-AR–mediated relaxant effect.
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Enhanced Contractile Response By PI3K Inhibition Is Associated With Increased Responses of ICa and Intracellular Ca2+ Transient to ß1-AR stimulation
Because L-type Ca2+ current (ICa) is the key factor of cardiac excitation-contraction coupling, we next determined whether it is involved in the inhibitory effect of PI3K on ß1-AR contractile response. Similar to the contractile response, ICa response to ß1-AR stimulation by NE (5x10–8 mol/L, in the presence of prazosin and ICI 118,551) was clearly augmented by the PI3K inhibitor, LY294002 (Figure 6A and 6B). On average, LY294002 enhanced the ß1-AR–stimulated current by 22.4±3.3% (n=9 cells; P<0.01). These results suggest that PI3K signaling offsets ß1-AR positive inotropic effect likely via inhibiting ß1-AR–induced Ca2+ influx through L-type Ca2+ channels. Notably, inhibition of PI3K exhibits a very minor effect on ß2-AR–induced increase in ICa (data not shown).
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To further delineate the mechanism underlying the potentiating effect of LY294002, myocyte contraction and Cai transient were simultaneously measured in indo1-loaded myocytes. Both parameters were examined in response to positive inotropic agents at a concentration close to EC50, including ISO (10–9 mol/L), NE (5x10–8 mol/L, plus prazosin and ICI 118,551), or FSK (5x10–7 mol/L), in the presence or absence of LY294002 (5x10–6 mol/L). As shown in Figure 7, the LY294002-induced increase in contraction amplitude in response to the aforementioned agonists was accompanied by a proportional increase in Cai transient, without altering its baseline (data not shown). These results indicate that PI3K antiadrenergic effect is mainly mediated by blunting ß1-AR/PKA–dependent increase in ICa and the consequent augmentation in the Cai transient, instead of altering myofilament response to intracellular Ca2+. This conclusion is further supported by the fact that LY294002 could not enhance the high extracellular [Ca2+]-induced positive inotropic effect (Figure 4).
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P<0.001 vs no LY. Baseline contraction amplitude is 5.75±0.46% (n=33 cells), whereas the basal Cai transient is 0.161±0.008 (n=33 cells).
ßARK1 As Well As Gß Signaling Is Involved in ß1-AR–Mediated PI3K Activation
Disruption of Gi signaling with PTX cannot block the potentiating effect of LY294002 on ß1-AR contractile response (Figure 8A), indicating that Gi is not involved in ß1-AR–mediated PI3K activation. In contrast, infection of cells with an adenovirus expressing ßARK-ct (a peptide inhibitor of ßARK117 as well as Gß signaling18) completely abolished the potentiating effect of the PI3K inhibitor (Figure 8B). In this subset of experiments, a lower concentration (10–8 mol/L) of NE was chosen to avoid possible saturation of cell contractile response. These data suggest that either Gß dissociated from Gs or ßARK1 plays an essential role in ß1-AR–induced PI3K signaling. To distinguish these two possibilities, we next examined the possible effects of LY294002 on FSK-induced contractile response in the presence or absence of ßARK-ct, and found that the potentiating effect of LY294002 on FSK response was also abolished by ßARK-ct (Figure 8B), indicating that ßARK1 is required for the function of PI3K in inhibiting cAMP/PKA-mediated contractile response in cardiomyocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both cAMP- and Gß
-ßARK1–Dependent Signaling Pathways Are Likely Involved in ß1-AR–Induced PI3K ActivationThe present study provides biochemical and physiological evidence that ß1-AR is able to increase PI3K activity, which in turn, evokes a negative regulation of the receptor-mediated increases in ICa and Cai transients, thus offsetting the receptor-induced positive inotropic effect. Disruption of Gi signaling by PTX cannot block the potentiating effect of LY294002 on ß1-AR contractile response. In contrast, ßARK-ct blocks the PI3K inhibitor–induced potentiating effect, indicating that either ßARK1 or the ß
heterodimer dissociated from Gs plays an essential role in PI3K-mediated negative regulation of ß1-AR contractile response. Interestingly, we have found that ßARK-ct fully abolishes the potentiating effect of LY294002 on FSK-mediated contractile response. These results indicate that ßARK1 is important for the normal functionality of PI3K, and plays an essential role in PI3K-mediated inhibition of cAMP-, as well as ß1-AR–, mediated positive contractile response. This conclusion is based on following lines of evidence. ßARK-ct, a well-characterized inhibitor of ßARK117 in addition to blocking Gß signaling,18 fully blocks the ability of LY294002 to enhance the contractile response to FSK. Because FSK activates the cAMP-PKA pathway via stimulation of AC in the absence of increased free Gß, the basal ßARK1 activation might be required for the function of PI3K in inhibiting cAMP/PKA-mediated contractile response in cardiomyocytes. This possibility is corroborated by the fact that PI3K and ßARK1 can form a tight physical complex in cardiac myocytes.19
Because Gß signaling is necessary for ßARK1 activation,20 the present results cannot rule out the possible involvement of Gß in ß1-AR–induced activation of PI3K. In this regard, previous studies have shown that the class IB PI3K (PI3K isoform) is a downstream target of Gß signaling.21,22 Recent studies, however, suggest that stimulation of ß-AR activates PI3K and ß in cultured neonatal rat cardiac myocytes.23 Regardless of the specific isoform of PI3Ks responding to ß1-AR stimulation, the present results indicate that ß1-AR stimulation markedly increases PI3K activation via a signaling mechanism involving Gß and Gß-activated ßARK1 in cardiomyocytes.
In addition to increased free Gß subunits and the subsequent activation of ßARK1, the Gs-AC-cAMP-PKA signaling cascade may also contribute to ß1-AR–induced PI3K activation, because direct activation of AC by FSK induces a robust increase in PI3K activity and because inhibition of PI3K selectively enhances the positive inotropic effect induced by FSK but not by high extracellular [Ca2+]. Thus, it is likely that the Gs-AC-cAMP-PKA, the free Gß, and ßARK1 participate in the receptor-evoked PI3K signaling, which leads to negative regulation of ß1-AR contractile response in cardiomyocytes.
PI3Ks Activated by ß1-AR Stimulation Differ From That Activated by ß2-AR Stimulation in Regulating Cardiac Excitation-Contraction Machinery and Myocyte Survival
Accumulating evidence indicates that coexisting cardiac ß-AR subtypes, mainly ß1-AR and ß2-AR, activate different signaling cascades with ß1-AR coupling to the classic Gs-PKA pathway and ß2-AR to concomitant Gs and Gi cascades.24–26 The coupling of ß2-AR to Gi proteins compartmentalizes the Gs-mediated cAMP signaling, resulting in specific modulation of L-type Ca2+ channels without affecting other intracellular regulatory proteins.11,12,24–26 Inhibition of PI3K, similar to PTX treatment, enables ß2-AR stimulation to induce a marked increase in phosphorylation of PLB at the PKA site, Ser,16 thus enabling ß2-AR stimulation to cause a de novo relaxant response and a markedly enhanced positive inotropic response in adult rat cardiomyocytes.7 Thus, PI3K is a key downstream event of acute ß2-AR/Gi signaling that negates the Gs-mediated cAMP signaling.
Notably, PI3Ks activated by ß1-AR stimulation distinctly differ from that activated by ß2-AR stimulation in many important ways. ß1-AR–evoked PI3K signaling is unable to suppress the receptor-induced PLB phosphorylation. To the contrary, ß1-AR– but not ß2-AR–activated PI3Ks offset the receptor-mediated increase in ICa. These results underscore that PI3Ks following different ß-AR subtype stimulation exhibit different selectivity for target proteins, although both ß-AR subtypes increase total PI3K activity to a similar extent and their positive inotropic effects are negatively regulated by PI3K signaling.
In addition to regulating cardiac excitation-contraction coupling, ß1-AR and ß2-AR cause opposing effects on cardiomyocyte survival and death. Whereas ß2-AR protects myocytes against apoptosis, ß1-AR promotes myocyte apoptotic death.5,6,27,28 These contrasting effects of ß-AR subtypes were initially explained by the ß2-AR–coupled Gi-Gß-PI3K signaling pathway.6 However, the present study has documented that ß1-AR stimulation is equally efficient in activating PI3Ks in these cardiomyocytes. It awaits further investigation to determine why ß1-AR–activated PI3K signaling, unlike that of ß2-AR, does not protect cardiomyocytes.
The exact mechanism underlying the differences between ß1-AR– and ß2-AR–activated PI3Ks in their target protein selectivity and in regulating myocyte survival and death remains largely elusive. Several candidate mechanisms might be involved. First, ß1-AR and ß2-AR might stimulate different PI3K isoforms. In this regard, it has been shown that different PI3K isoforms exhibit distinct functional roles.29 Second, the differential interaction of ß-AR subtypes with PI3Ks might be, in part, attributed to the distinct subcellular distribution of these ß-AR subtypes,30,31 therefore accessing distinct downstream signaling pathways. In addition, stimulation of ß2-AR, but not ß1-AR, activates PI3Ks in a PTX-sensitive manner.7 Thus, distinct G protein coupling and subcellular localization of ß1-AR and ß2-AR may render the subtype-specific ß-AR/PI3K interaction in regulating cardiac excitation-contraction coupling and cell survival.
Physiological and Pathophysiological Relevance
In many biological systems, stimulation of a GPCR by agonists rapidly blunts the receptor signaling efficiency (receptor desensitization). It has been well established that ßARK1 plays an essential role in agonist-initiated ß-AR desensitization by phosphorylation of activated ß-ARs both in vitro and in vivo.17,32 Because PI3K is able to physically associate with ßARK1 and promotes agonist-dependent ß-AR internalization,19 activation of PI3K may be involved in agonist-dependent ß-AR desensitization. The inhibitory effect of PI3K on the ß1-AR–mediated contractile response may explain, at least in part, agonist-dependent desensitization of the receptor. Indeed, the present results indicate that PI3K is a key downstream event of acute ß1-AR–Gs-Gß signaling and subsequent ßARK1 activation that negates the receptor-mediated cAMP signaling in terms of changes in cardiac ICa and contractility.
ß1-AR signaling can be modulated by other GPCRs expressed in cardiac sarcolemmal membranes, including PTX-sensitive Gi-coupled adenosine receptors,33 acetylcholine receptors,34 or opioid receptors.35,36 For example, activation of M2 acetycholine receptors increases PI3K activity in rat neonatal myocytes.5 The counteraction of PI3K signaling on ß1-AR–mediated contractile response raises an important question whether PI3K signaling plays a role in the antiadrenergic effect of the aforementioned PTX-sensitive GPCRs. Our data have further revealed that ßARK1 activation plays an essential role in PKA-evoked PI3K signaling. These unexpected findings suggest that PKA might elicit ßARK1 activation, or alternatively, the basal activation of ßARK1 is necessary for the function of PI3K in inhibiting PKA-mediated contractile response. Altogether, these observations imply that PI3K activation may be generally involved in regulating multiple signal transduction cascades in cardiomyocytes, perhaps in many other cell types as well, and that the present study may only demonstrate some important facets of PI3K-mediated regulation of cardiac performance.
The interaction between PI3K and ß1-AR-Gs-PKA in regulating cardiomyocyte ICa and contractility may also have important pathophysiological relevance. In this regard, it has been shown that enhanced PI3K activity plays essential roles in pressure overload-induced cardiac hypertrophy,37 ß-AR–mediated hypertrophy, and hypertrophy marker gene expression.23,38 In light of its role in pathological cardiac hypertrophy and its negative inotropic effect, exaggerated PI3K activation might be implicated in the overall process of cardiac remodeling and chronic heart failure. This perception has been corroborated by the recent notion that disruption of the association of PI3K to agonist-activated ß-AR prevents catecholamine-induced ß-AR downregulation and ameliorates the development of heart failure in response to pressure overload.39 Thus, the inhibitory effect of PI3K on ß1-AR–mediated positive inotropic response contributes to the pathogenesis of heart failure, and inhibition of the interaction of the receptor and PI3K might open a novel therapeutic avenue to restore ß-AR signaling and improves the function of the failing heart. Additionally, recent studies have suggested a heart failure-associated upregulation of ß3-ARs and alteration of its negative inotropic effect.40,41 It merits future investigation to determine the potential role of PI3K signaling in ß3-AR–mediated negative inotropic effect, particularly in the failing heart.
In summary, ß1-AR stimulation elevates PI3K activity likely via both the Gs- and the Gsß-ßARK1-directed signaling pathways in adult rat cardiomyocytes. Enhanced PI3K signaling negatively regulates ß1-AR–induced contractile response, mainly via interfering with the receptor-mediated increases in ICa and intracellular Ca2+ transients, without altering myofilament Ca2+ response or the receptor-mediated phosphorylation of PLB. The PI3K-induced inhibition of ß1-AR contractile response might be involved in agonist-induced desensitization of the receptor, and when exaggerated, contributes to the pathogenesis of heart failure.
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Acknowledgments |
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This work is supported by NIH intramural research grant (to L.V., S.H.J., V.M., M.T.C., E.G.L., and R.P.X.) and, in part, by Chinese National Natural Science Foundation (30100215), Peking University 985 Project, Chinese National Key Project 973 (G2000056906), and Chinese Young Investigator Award (30225036). We thank Dr H. Cheng for critical comments and discussions, and Dr H. Spurgeon and B. Ziman for their excellent technique support.
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Footnotes |
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This manuscript was sent to Hans Michael Piper, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
*These authors contributed equally to this article. 
Original received June 3, 2004; revision received October 18, 2004; accepted October 28, 2004.
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