Published online before print May 16, 2005, doi: 10.1161/CIRCULATIONAHA.104.508796
(Circulation. 2005;111:2579-2587.)
© 2005 American Heart Association, Inc.
Heart Failure |
Restoration of ß-Adrenergic Receptor Signaling and Contractile Function in Heart Failure by Disruption of the ßARK1/Phosphoinositide 3-Kinase Complex
From the Department of Medicine, Cell Biology and Molecular Genetics (C.P., S.V.N.P., H.A.R.), and Department of Surgery (J.N.S., J.A.H., C.M.), Duke University Medical Center, Durham, NC.
Correspondence to Howard A. Rockman, MD, Department of Medicine, Cell Biology and Molecular Genetics, DUMC 3104, Room 226, CARL Building, Durham, NC 27710. E-mail rockm001{at}mc.duke.edu
Received September 22, 2004; revision received January 10, 2005; accepted January 13, 2005.
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Abstract |
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Background— Desensitization and downregulation of myocardial ß-adrenergic receptors (ßARs) are initiated by the increase in ßAR kinase 1 (ßARK1) levels. By interacting with ßARK1 through the phosphoinositide kinase (PIK) domain, phosphoinositide 3-kinase (PI3K) is targeted to agonist-stimulated ßARs, where it regulates endocytosis. We tested the hypothesis that inhibition of receptor-targeted PI3K activity would alter receptor trafficking and ameliorate ßAR signaling, ultimately improving contractility of failing cardiomyocytes.
Methods and Results— To competitively displace PI3K from ßARK1, we generated mice with cardiac-specific overexpression of the PIK domain. Seven-day isoproterenol administration in wild-type mice induced desensitization of ßARs and their redistribution from the plasma membrane to early and late endosomes. In contrast, transgenic PIK overexpression prevented the redistribution of ßARs away from the plasma membrane and preserved their responsiveness to agonist. We further tested whether PIK overexpression could normalize already established ßAR abnormalities and ameliorate contractile dysfunction in a large animal model of heart failure induced by rapid ventricular pacing in pigs. Failing porcine hearts showed increased ßARK1-associated PI3K activity and marked desensitization and redistribution of ßARs to endosomal compartments. Importantly, adenoviral gene transfer of the PIK domain in failing pig myocytes resulted in reduced receptor-localized PI3K activity and restored to nearly normal agonist-stimulated cardiomyocyte contractility.
Conclusions— These data indicate that the heart failure state is associated with a maladaptive redistribution of ßARs away from the plasma membrane that can be counteracted through a strategy that targets the ßARK1/PI3K complex.
Key Words: catecholamines • gene therapy • heart failure • receptors, adrenergic, beta
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Introduction |
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Abnormalities in the ß-adrenergic receptor (ßAR) signaling system such as a reduction in the number of ligand-accessible ßARs (downregulation) and diminished response to catecholamine stimulation of remaining receptors (desensitization) are hallmarks of heart failure.1,2 However, whether changes in ßAR signaling represent an adaptive and protective process, as some postulate,3 or whether ßAR dysregulation actually promotes deterioration of cardiac function4 is still controversial.3 Results from our previous studies suggest that chronic ßAR dysfunction in the failing heart is maladaptive and contributes to the deterioration in cardiac function.4 Indeed, a consistent and prominent feature of ß-blocker therapy in heart failure is the reversal of ßAR dysfunction.5,6
Considerable evidence supports the concept that the chronic increase in circulating catecholamine levels is largely responsible for the ßAR abnormalities found in failing hearts.4 Agonist-induced receptor dysfunction begins with ßAR phosphorylation by ßARK1, followed by ß-arrestin binding that sterically interdicts further G-protein coupling and initiates the process of receptor internalization.4 Once internalized, receptors are targeted to specialized intracellular compartments, where they can be dephosphorylated and recycled to the plasma membrane (early endosomes) or sent to the degradation pathway (late endosomes).4 Interestingly, accumulating evidence suggests that the process of ßAR internalization per se may be pathological because internalizing receptors can directly activate maladaptive signaling pathways in a G-protein–independent fashion.7 Therefore, strategies that might prevent this redistribution may exert a beneficial effect in heart failure. At the present time, very little is known about the intracellular fate of internalized ßARs in heart failure and, more importantly, about the signaling pathways potentially involved in the resensitization and recycling to the plasma membrane of these vesicular pools of ßARs.
We have recently shown that efficient ßAR internalization requires the recruitment of phosphoinositide 3-kinase (PI3K) to agonist-stimulated ßARs.8,9 This process depends on the cytosolic association of PI3K with ßARK1 through the helical domain of PI3K, also known as the phosphoinositide kinase (PIK) domain.8,9 Inhibition of ßAR-localized PI3K activity by transgenic overexpression of an inactive form of PI3K
(PI3Kinact) prevents the development of ßAR dysfunction in response to both chronic catecholamine stimulation and pressure overload in vivo.10 Interestingly, our previous in vitro studies have shown that overexpression of the minimal 197 aa PIK domain displaces endogenous PI3K from ßARK1, thereby interfering with agonist-stimulated ßAR internalization.9
From these data, we hypothesized that disruption of the ßARK1/PI3K complex through transgenic overexpression of the PIK domain would preserve ßAR signaling in vivo under conditions of chronic catecholamine stimulation. Furthermore, we used an adenoviral gene transfer approach to determine whether overexpression of the PIK domain peptide under conditions of established ßAR dysfunction would restore the contractile responsiveness to ß-agonist stimulation in failing cardiomyocytes.
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Methods |
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Generation of Transgenic Mice
The FLAG-tagged PIK domain9 was directionally subcloned into a vector downstream of
-myosin heavy chain (MHC) gene promoter and upstream of the SV40 polyadenylation site. Transgenic founders were identified by Southern blot analysis of tail DNA using the SV40 poly (A) as a probe. Transgenic founder mice were backcrossed into a C57BL/6 background for 7 generations before being used in experiments to investigate the phenotype. Western blot analysis was carried out on multiple generations to analyze and confirm high transgene expression. Animals were handled according to the approved protocols and animal welfare regulations of the Institutional Review Board at Duke University Medical Center.
Membrane Fractionation, Lipid Kinase Assay, ßARs Radioligand Binding, and Adenylyl Cyclase Activity
Plasma membrane and cytosolic fractions from left ventricles flash-frozen in liquid N2 were separated by centrifugation at 37 000g as previously described.11 Lipid kinase assays on cytosolic fractions were performed after immunoprecipitation with antibodies directed against pan-PI3K and PI3K and isoforms (Santa Cruz) as previously described.8 Then, 400 µg of plasma membrane fractions was used for immunoprecipitation with a polyclonal antibody directed against ßARK1 (Santa Cruz) to measure ßARK1-associated PI3K activity. Early and late endosomal fractions were recovered after ultracentrifugation of the crude cytosolic fraction for 1 hour at 300 000g and 200 000g, respectively. Receptor binding with 20 µg of protein from the plasma membrane and early/late endosomal fractions was performed as described previously using ßAR ligand [125I] cyanopindolol (250 pmol/L).11 Adenylyl cyclase assays were performed as described previously,11 using 20 µg of the plasma membrane fraction.
Generation of Recombinant Adenoviruses to Overexpress FLAG-PIK
To generate adenoviruses directing the expression of the PIK domain of PI3K, cDNA-encoding FLAG-tagged PIK domain9 was amplified to introduce convenient restriction enzymes sites XhoI and HindIII (forward, 5-CTCGAGCCGCCGCCGCGGATAGCCCTCCTAAG-3; reverse: 5-AAGCTTCTAGTCGTGCAGCAT-3). Amplified fragments were digested with XhoI and HindIII enzymes and ligated into pShuttle-CMV. Recombinant pShuttle-CMV was coelectroporated with pAdEasy-1 into BJ5183 Escherichia coli (Stratagene) to generate recombinant adenoviral vector.12 The recombinant adenoviral DNA was transfected into human embryonic kidney 293 cells using Lipofectamine (Invitrogen), and the viruses were serially amplified and purified on a CsCl density gradient by ultracentrifugation. Control adenoviruses consisting of the identical adenovirus backbone without the cDNA insert ("empty virus," AdEV) or with a GFP insert (AdGFP) were kindly provided by Dr Christopher J. Kontos (Duke University Medical Center, Durham, NC) for amplification.
Expanded Methods can be found in the online-only Data Supplement.
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Results |
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Mice With Cardiac-Specific Overexpression of PIK Domain Have Reduced ßARK1-Associated PI3K Activity
To disrupt the ßARK1/PI3K complex in vivo, we generated transgenic mice with cardiac-specific overexpression of the PIK domain peptide of PI3K using the
MHC promoter (Figure 1a, top).13 Three founders (11, 26, and 14) were generated that had 16-, 108-, and 198-fold overexpression of the transgene relative to wild-type (WT) mice (Figure 1a, bottom). Because we have previously shown that the PIK domain competitively displaces PI3K from ßARK1 in a concentration-dependent manner,9 we used the 198-fold overexpressing mice to determine whether the PIK transgenic mice had decreased ßARK1-associated PI3K activity. ßARK1 was immunoprecipitated from left ventricular lysates and assayed for associated PI3K activity. Compared with WT littermates, PIK-overexpressing transgenic mice (TgPIK) showed a significant reduction in ßARK1-associated PI3K (Figure 1b and 1c) as a result of the displacement of endogenous PI3K from ßARK1 (Figure 1b). Next, we tested whether overexpression of the PIK domain in transgenic mice altered total PI3K activity in the heart. PI3K and PI3K were immunoprecipitated from left ventricular lysates and assayed for lipid kinase activity. As expected, no difference in PI3K activity was observed between the PIK transgenic mice and their littermate controls (CON; Figure 1d). Importantly, these studies show that overexpression of the PIK domain specifically displaces endogenous PI3K from the ßARK1 complex but does not affect endogenous PI3K activity.
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Cardiac-Specific Overexpression of the PIK Domain Prevents Isoproterenol-Induced ßAR Desensitization and Downregulation In Vivo
We next tested whether overexpression of PIK domain peptide would prevent ßAR dysfunction in vivo after long-term exposure to high levels of catecholamines. TgPIK mice (198-fold) and their WT littermates were chronically treated with the catecholamine isoproterenol (ISO) for 7 days via osmotic mini-pumps. ISO administration for 7 days determined a similar mild hypertrophic response in WT and TgPIK mice (Figure 2a, black bars). To test the effects of PIK overexpression on ßAR levels at the plasma membrane, ßAR density was directly measured in myocardial plasma membrane fractions from WT and transgenic mouse hearts after 7 days of chronic ISO treatment (Figure 2b). Plasma membrane ßAR density was significantly reduced by 40% in chronic ISO-treated WT hearts compared with vehicle-treated WT hearts, whereas no significant decrease in ßAR density occurred in the ISO-treated TgPIK hearts compared with vehicle-treated TgPIK hearts (Figure 2b, black bars). To precisely determine the intracellular fate of chronically stimulated receptors in WT and TgPIK hearts, ultracentrifugation was carried out to separate the vesicular fractions corresponding to the early and late endosomal compartments. Isolation of early and late endosomes was confirmed by immunoblotting with antibodies against rab5 and lamp1 proteins, markers for early and late endosomes, respectively (Figure 2c). Interestingly, chronic stimulation of WT mice with ISO led to a significant redistribution of ßARs into the early and late endosomal fractions (Figure 2, d-e). In sharp contrast, in PIK-overexpressing mouse hearts exposed to chronic catecholamine stimulation, ßARs showed no redistribution into endosomal fractions (Figure 2d and 2e). Importantly, no differences were observed in total receptor number among the different groups (Figure 2f, black and white bars). Moreover, the level of steady-state mRNA expression for ß1ARs was unchanged in the different groups (Data Supplement, Figure A). These data indicate that the agonist-promoted downregulation of ßARs at the plasma membrane actually results in a redistribution of receptors into specialized intracellular compartments, with enrichment into early and late endosomal organelles and preservation of the total cellular receptor number.
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Because disruption of the ßARK1/PI3K complex by PIK overexpression maintains ßAR levels at the plasma membrane without inhibiting agonist-induced ßAR phosphorylation and desensitization,12 we tested whether increased numbers of agonist-accessible ßARs would result in an increase in downstream signaling. To test this, we measured basal and ISO-stimulated cAMP generation in membranes from WT and TgPIK hearts after 7 days of chronic ISO treatment (Figure 2g). As expected, no differences were found for basal cAMP production among all the different groups (Figure 2g, white bars), and a significant reduction in ISO-stimulated cAMP was observed in WT mice treated with catecholamines. Importantly, overexpression of the PIK domain completely preserved adenylyl cyclase activity after chronic ISO compared with WT (Figure 2g, black bars). These data indicate that PIK overexpression not only maintains membrane levels of ßARs but also promotes their resensitization despite prolonged agonist stimulation.
Overexpression of PIK domain exerted these beneficial effects without affecting other downstream PI3K signaling pathways, as measured by similar activation of protein kinase B (PKB) and glycogen synthase kinase (GSK) in WT and TgPIK mice (Figure 2h), as previously described.9 Moreover, overexpression of the PIK domain in transgenic mice did not alter the low rate of apoptotic cell death induced by prolonged ISO stimulation (Data Supplement, Figure B). Taken together, these results indicate that disruption of the ßARK1/PI3K in the heart preserves in vivo ßAR signaling under conditions of increased circulating catecholamines through selective and receptor-targeted inhibition of PI3K activity. Interestingly, PIK overexpression depletes early and late endosomes of ßARs despite prolonged agonist stimulation, maintaining their levels at the plasma membrane and restoring their ability to signal.
To confirm whether PIK overexpression would preserve cardiac function under pathological conditions, similar to what we have shown in mice expressing a kinase-dead mutant PI3K (PI3Kinact),10 we performed transverse aortic constriction experiments in WT and TgPIK mice. Consistent with our previous report,10 pressure-overloaded TgPIK mice displayed significantly improved cardiac function after 4 weeks of pressure overload compared with WT mice despite the presence of similar trans-stenotic pressure gradients (Data Supplement, Figure C).
Increased ßARK1-Associated PI3K Activity Results in Profound Abnormalities of ßAR Signaling in Porcine Heart Failure
The previous results in ISO-treated WT mice indicate that prolonged agonist stimulation promotes a shift of ßARs from the plasma membrane to specialized intracellular vesicular compartments, from where they can undergo de-phosphorylation and recycling (early endosomes)14 or degradation (late endosomes).15 PIK overexpression inhibits this process in mice and facilitates receptor re-sensitization despite prolonged agonist stimulation. Since these observations have important clinical implications in terms of restoring ßAR signaling in the failing heart, we tested whether intracellular redistribution of ßARs occurs in experimental heart failure and can be reversed by PIK overexpression. Therefore, we characterized PI3K signaling and ßAR abnormalities in a large animal model of cardiac dysfunction induced by rapid ventricular pacing in pigs,16 which closely recapitulates the bio-mechanical complexity of human heart failure. After two weeks of pacing-induced tachycardia, pigs developed a reproducible phenotype of dilated cardiomyopathy, characterized by marked cardiac enlargement and dysfunction, with the pacer on or off (Table 1). Cardiac dysfunction in failing pig hearts was accompanied by a marked increase in ßARK1 protein levels (Figure 3a, top panel) and ßARK1-associated PI3K activity (Figure 3a, middle and lower panels). The increase in ßARK1-associated PI3K activity in the plasma membrane was associated with a significant and selective increase in the activity of PI3K isoform (Figure 3), while no appreciable increase in PI3K activity was observed (Data Supplement, Figure D), consistent with our previous data with pressure overload-induced heart failure in mice.17
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P<0.01, HF vs CON. HC indicates heavy chain of IgG.Increased ßARK1 levels and membrane-targeted PI3K activity in failing pig heart were associated with profound abnormalities in ßAR signaling, similar to those described in human heart failure1,2 and in our model of ßAR dysfunction induced by chronic catecholamine stimulation. ßARs were significantly desensitized, as expressed by diminished ISO-stimulated membrane adenylyl cyclase activity, either absolute (Figure 4a) or normalized as percent maximal activity induced by sodium fluoride (NaF) (Figure 4b). Moreover, ßAR plasma membrane levels were significantly reduced by 25% in the heart failure (HF) group compared with the CON group (Figure 4c; CON, 156.9±7.6 fmol/mg; HF, 118.2±10.7 fmol/mg; P<0.01). Importantly, HF hearts displayed redistribution of ßARs in both early and late endosomal fractions compared with CON (Figure 4d), showing that the sequestration of ßARs into specialized intracellular compartments takes place in this large animal model of heart failure.
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Adenovirus-Mediated Overexpression of PIK Domain Restores Contractile Function of Cardiac Myocytes Isolated From Failing Pig Hearts
To test the ability of PIK overexpression to reverse the established ßAR abnormalities in failing pig hearts, we generated recombinant adenoviruses driving the expression of FLAG-tagged PIK domain of PI3K. In cardiac myocytes from porcine hearts, infection at a multiplicity of 1000 resulted in significant expression of PIK domain peptide, which was confirmed by immunofluorescence (Figure 5a) and immunoblotting (Figure 5b).
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Because failing pig hearts display profound ßAR abnormalities with an associated increase in ßARK1-associated PI3K activity (Figures 3 and 4
), we tested whether adenovirus-mediated overexpression of the PIK (AdPIK) domain could displace endogenous PI3K from ßARK1 in failing cardiomyocytes. Thirty-six to 48 hours after infection, membrane fractions from primary cultures of porcine cardiac myocytes were used for ßARK1-associated PI3K assay. As shown in Figure 5c, we observed a marked decrease in receptor-localized PI3K activity in the cardiac cells infected with AdPIK compared with uninfected cells or cells infected with the viral backbone (AdEV) (Figure 5c), showing that overexpression of PIK domain peptide in myocytes displaced endogenous PI3K from ßARK1.
To test the effects of adenovirus-driven overexpression of PIK on receptor endocytosis induced by agonist binding, ß2AR internalization after ISO stimulation was visualized in sarcoma cells by confocal microscopy. Cells transiently expressing ß2AR-YFP were infected with either AdEV or AdPIK (infection at a multiplicity of 100). In the absence of agonist, a distinct membrane localization of ß2AR-YFP was visualized in uninfected and infected cells (Figure 5d, panels 1 through 3). After ISO stimulation, receptors were completely redistributed into cellular aggregates in uninfected cells and cells infected with AdEV (Figure 5d, panels 5 and 6). In contrast, in cells overexpressing the PIK domain peptide at high levels, as visualized by Texas red staining, there was complete preservation of ß2AR-YFP membrane staining (Figure 5d, panels 7 and 8). Importantly, PIK overexpression similarly blocks ß1AR internalization after agonist stimulation (Data Supplement, Figure E). These data confirm that the net effect of PIK overexpression is to preserve ßAR levels on the plasma membrane despite agonist stimulation.
To test whether PIK overexpression could rapidly reconstitute ßAR responsiveness to agonist in failing cardiomyocytes, contractility studies were carried out on cells isolated from CON and HF pig hearts, uninfected (HF) or at 36 to 48 hours after infection with either empty virus AdEV (HF-AdEV) or AdPIK (HF-AdPIK). In multiple sets of cells, we measured basal and ISO- stimulated percent cell shortening, velocity of shortening, and velocity of relaxation. Failing pig myocytes were characterized by marked depression of all the indices of contractility compared with cells from normal pig hearts (Table 2). Importantly, infection with AdPIK markedly ameliorated the abnormalities in ISO-stimulated cellular contractility compared with AdEV (Figure 6), reversing the contractility parameters to almost normal levels (Table 2). Therefore, disruption of the ßARK1/PI3K complex improves failing myocyte function by reversing already established abnormalities in ßAR function and thereby restores normal responsiveness to ßAR agonist stimulation (Table 2 and Figure 6). The limited amount of material from the primary cultures of pig cardiomyocytes after AdPIK or AdEV infection did not allow us to directly evaluate the levels of ßARs or cAMP generation in these cells. However, restoration of ßAR responsiveness after inhibition of ßARK1-associated PI3K activity is consistent with a process that involves reconstitution of functional ßARs at the plasma membrane. Taken together, these results indicate that PIK overexpression changes the fate of agonist-stimulated ßARs, preserving ßAR signaling despite prolonged catecholamine administration and, importantly, reversing already established ßAR abnormalities, thereby restoring contractility of failing cardiomyocytes to nearly normal values.
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10 different cells per heart. *P<0.01, all ISO vs basal; |
Discussion |
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We show in the present study that the membrane targeting of PI3K plays a major role in the processes of ßAR desensitization and downregulation that are characteristic of heart failure. Chronic catecholamine stimulation and heart failure lead to a loss of ßARs on the plasma membrane and a redistribution of receptors to endosomal compartments. Disruption of the ßARK1/PI3K complex by overexpressing the 197 aa PIK domain peptide prevents endogenous PI3K translocation to the membrane and alters the intracellular trafficking of ßARs after prolonged agonist stimulation. The resulting effect is to preserve ßAR membrane levels and their ability to signal. Importantly, overexpression of the PIK domain peptide in failing cardiomyocytes can also reverse already established ßAR abnormalities, reconstituting to nearly normal the contractile response to ß-agonist stimulation.
Human heart failure is characterized by increased levels of circulating catecholamines and extensive abnormalities in ßAR signaling that are due in part to an increase in ßARK1 levels.1,2,18 Data from our present study and from previous studies have always suggested that chronic ßAR dysfunction promotes deterioration of cardiac function; therefore, strategies that restore ßAR signaling may represent a valuable strategy in the treatment of heart failure.4 This conclusion is supported by a number of observations in which preservation of ßAR signaling by ßARK1 inhibition,19,20 PI3Kinact overexpression,10 or PIK domain overexpression as in the present study results in the amelioration of cardiac function10,19,20 and prolongation of survival.9 These results are congruent with data showing the reversal of ßAR dysfunction after ß-blocker therapy in heart failure.5,6 Importantly, the concept of normalizing ßAR dysfunction in HF needs to be distinguished from strategies that use chronic ßAR activation as a therapy such as chronic catecholamine infusion,21 overexpression of ßARs,22,23 or Gs.24 All of these approaches share a common feature—chronic ßAR activation—that leads to internalization and downregulation of receptors, a process we believe is inherently maladaptive. Our data using different genetically modified animal models, including the present study, consistently support the concept that restoration of normal responsiveness to agonist rather than chronic receptor activation is required to exert a beneficial effect in heart failure.10,19,20
The earliest processes that ultimately lead to the dampening of ßAR signaling are ßAR phosphorylation by ßARK1, binding of ß-arrestin, and targeting of the agonist-bound receptor to endocytosis.4 According to the current understanding, after internalization, ßARs are targeted to endosomal compartments where they can be either dephosphorylated and recycled back to the plasma membrane to start a fresh cycle of signaling (early endosomes) or targeted to the late endosomes and degraded. Taken together, our results suggest a new paradigm for ßAR redistribution from the plasma membrane to intracellular compartments. Indeed, although we find a significant accumulation of receptors into early and late endosomes, we unexpectedly find that the total number of ßARs and their mRNA levels remain constant after ISO stimulation. Although it is not known for how long and to what extent the sequestered receptors reside in late endosomes before degradation, our data suggest that these processes in vivo are not rapid. Therefore, our data suggest that desensitized and internalized receptors can be targeted to rapidly recycle back to the plasma membrane from early and possibly late endosomes. A strategy such as PIK overexpression would achieve this goal, as we show in these studies.
A recent study suggests that a continuous equilibrium exists in vivo between internalization and recycling of ßARs to the sarcolemma under physiological conditions and that this plasma membrane–endosome bidirectional trafficking of ßARs may be responsible for the resensitization of receptors after catecholamine exposure.25 Our recent in vitro studies have shown that generation of phosphoinositides by PI3K at the site of activated receptors is required for efficient internalization of ßARs after agonist stimulation.9 Consistently, in pigs with pacing-induced heart failure, ßAR redistribution to intracellular compartments was accompanied by a significant increase in membrane-targeted PI3K activity. Although our data in failing porcine hearts suggest a specific role for PI3K in the pathophysiology of heart failure, we have previously shown10 that the loss of PI3K is insufficient to prevent ßAR abnormalities after chronic catecholamine stimulation,10 because ßARK1 can associate with and recruit other isoforms of PI3K to the receptor complex.9,10 Consistent with our previous studies,10 it has recently been reported that PI3K KO mice undergo deterioration in cardiac function after pressure overload with necrosis and fibrosis, possibly because of hyperstimulation of ßARs, because treatment with the ßAR antagonist propranolol limited these adverse affects.26 These results support our concept that to normalize ßAR signaling, displacement of all PI3K isoforms from ßARK1, rather than inhibition of a specific isoform, is required. This is achieved by overexpression of the PIK domain, conserved throughout all isoforms of PI3K.
By disrupting the ßARK1/PI3K complex, which targets PI3K to the site of activated receptors, PIK overexpression reduces the amount of ßAR-targeted PI3K activity. Importantly, PIK overexpression in transgenic mice significantly altered the pattern of ßAR intracellular redistribution after agonist stimulation, with a resultant preservation of the plasma membrane levels of ßARs. Our data support an emerging concept that internalization of ßARs is a pathological process per se, because it directly activates maladaptive signaling pathways in a G-protein–independent fashion.7 Therefore, inhibition of ßAR redistribution accomplished by PIK overexpression is beneficial because it blocks maladaptive signaling pathways triggered by ßAR internalization. Interestingly, the significant increase in the ISO responsiveness of TgPIK hearts after chronic catecholamine stimulation suggests that overexpression of PIK domain peptide not only prevents receptor downregulation but also promotes receptor resensitization. Because PIK overexpression does not prevent ßARK1 membrane translocation and phosphorylation of ßARs,9 it is likely that the preservation of ßAR responsiveness in ISO-treated TgPIK mice occurs through enhanced rates of receptor dephosphorylation and resensitization. This concept is also supported by the evidence that adenovirus-mediated PIK overexpression under conditions of established desensitization and downregulation of ßARs still reconstitutes the contractile responsiveness of failing cardiomyocytes to ISO. Moreover, studies have shown that receptor dephosphorylation by protein phosphatase 2A requires the internalization of agonist-stimulated receptors.14 Taken together, these results suggest that PIK overexpression either promotes a rapid cycle of receptor/dephosphorylation and endosomal recycling or allows dephosphorylation at the plasma membrane. In both cases, the net effect is to preserve levels and function of agonist-accessible ßARs.
In conclusion, our studies show that targeting the ßARK1/PI3K complex with molecular interventions like overexpression of the PIK domain of PI3K, or of similar small molecules capable of disrupting the ßARK1/PI3K protein-protein interaction, represents a novel approach to restore ßAR function in heart failure. This would prevent the accumulation of ßARs within intracellular pools, preserving their plasma membrane levels and restoring their capability to properly signal without interfering with ßARK1 phosphorylation of activated receptors9 or with other PI3K downstream signaling pathways.
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Acknowledgments |
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This work was supported in part by NIH grants to Howard A. Rockman (HL–61558) and Carmelo Milano (HL–072183).
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Footnotes |
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*Drs Perrino and Naga Prasad contributed equally to this article.
The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.104.508796/DC1.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Bristow MR, Ginsburg R, Minobe W, Cubicciotti RS, Sageman WS, Lurie K, Billingham ME, Harrison DC, Stinson EB. Decreased catecholamine sensitivity and ß-adrenergic-receptor density in failing human hearts. N Engl J Med. 1982; 307: 205–211.[Abstract]
2. Bristow MR. Why does the myocardium fail? Insights from basic science. Lancet. 1998; 352 (suppl 1): SI8–SI14.[CrossRef][Medline] [Order article via Infotrieve]
3. Lohse MJ, Engelhardt S, Eschenhagen T. What is the role of ß-adrenergic signaling in heart failure? Circ Res. 2003; 93: 896–906.
4. Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. Nature. 2002; 415: 206–212.[CrossRef][Medline] [Order article via Infotrieve]
5. Heilbrunn SM, Shah P, Bristow MR, Valantine HA, Ginsburg R, Fowler MB. Increased ß-receptor density and improved hemodynamic response to catecholamine stimulation during long-term metoprolol therapy in heart failure from dilated cardiomyopathy. Circulation. 1989; 79: 483–490.
6. Reiken S, Wehrens XH, Vest JA, Barbone A, Klotz S, Mancini D, Burkhoff D, Marks AR. ß-Blockers restore calcium release channel function and improve cardiac muscle performance in human heart failure. Circulation. 2003; 107: 2459–2466.
7. Lefkowitz RJ, Whalen EJ. ß-Arrestins: traffic cops of cell signaling. Curr Opin Cell Biol. 2004; 16: 162–168.[CrossRef][Medline] [Order article via Infotrieve]
8. Naga Prasad SV, Barak LS, Rapacciuolo A, Caron MG, Rockman HA. Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1: a role in receptor sequestration. J Biol Chem. 2001; 276: 18953–18959.
9. Naga Prasad SV, Laporte SA, Chamberlain D, Caron MG, Barak L, Rockman HA. Phosphoinositide 3-kinase regulates ß2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/ß-arrestin complex. J Cell Biol. 2002; 158: 563–575.
10. Nienaber JJ, Tachibana H, Naga Prasad SV, Esposito G, Wu D, Mao L, Rockman HA. Inhibition of receptor-localized PI3K preserves cardiac beta-adrenergic receptor function and ameliorates pressure overload heart failure. J Clin Invest. 2003; 112: 1067–1079.[CrossRef][Medline] [Order article via Infotrieve]
11. Esposito G, Rapacciuolo A, Naga Prasad SV, Takaoka H, Thomas SA, Koch WJ, Rockman HA. Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation. 2002; 105: 85–92.
12. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A. 1998; 95: 2509–2514.
13. Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J. Tissue-specific regulation of the -myosin heavy chain gene promoter in transgenic mice. J Biol Chem. 1991; 266: 24613–24620.
14. Krueger KM, Daaka Y, Pitcher JA, Lefkowitz RJ. The role of sequestration in G protein-coupled receptor resensitization: regulation of ß2-adrenergic receptor dephosphorylation by vesicular acidification. J Biol Chem. 1997; 272: 5–8.
15. Whistler JL, Enquist J, Marley A, Fong J, Gladher F, Tsuruda P, Murray SR, Von Zastrow M. Modulation of postendocytic sorting of G protein-coupled receptors. Science. 2002; 297: 615–620.
16. Ping P, Gelzer-Bell R, Roth DA, Kiel D, Insel PA, Hammond HK. Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart. J Clin Invest. 1995; 95: 1271–1280.[Medline] [Order article via Infotrieve]
17. Naga Prasad SV, Esposito G, Mao L, Koch WJ, Rockman HA. Gß-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy. J Biol Chem. 2000; 275: 4693–4698.
18. Bristow MR, Minobe WA, Raynolds MV, Port JD, Rasmussen R, Ray PE, Feldman AM. Reduced ß1 receptor messenger RNA abundance in the failing human heart. J Clin Invest. 1993; 92: 2737–2745.[Medline] [Order article via Infotrieve]
19. Rockman HA, Chien KR, Choi DJ, Iaccarino G, Hunter JJ, Ross J Jr, Lefkowitz RJ, Koch WJ. Expression of a ß-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc Natl Acad Sci U S A. 1998; 95: 7000–7005.
20. Harding VB, Jones LR, Lefkowitz RJ, Koch WJ, Rockman HA. Cardiac ßARK1 inhibition prolongs survival and augments ß-blocker therapy in a mouse model of severe heart failure. Proc Natl Acad Sci U S A. 2001; 98: 5809–5814.
21. Remme WJ. Inodilator therapy for heart failure: early, late, or not at all? Circulation. 1993; 87 (suppl IV): IV-97–IV-107.[Medline] [Order article via Infotrieve]
22. Engelhardt S, Hein L, Wiesmann F, Lohse MJ. Progressive hypertrophy and heart failure in ß1-adrenergic receptor transgenic mice. Proc Natl Acad Sci U S A. 1999; 96: 7059–7064.
23. Bisognano JD, Weinberger HD, Bohlmeyer TJ, Pende A, Raynolds MV, Sastravaha A, Roden R, Asano K, Blaxall BC, Wu SC, Communal C, Singh K, Colucci W, Bristow MR, Port DJ. Myocardial-directed overexpression of the human ß(1)-adrenergic receptor in transgenic mice. J Mol Cell Cardiol. 2000; 32: 817–830.[CrossRef][Medline] [Order article via Infotrieve]
24. Iwase M, Bishop SP, Uechi M, Vatner DE, Shannon RP, Kudej RK, Wight DC, Wagner TE, Ishikawa Y, Homcy CJ, Vatner SF. Adverse effects of chronic endogenous sympathetic drive induced by cardiac GS overexpression. Circ Res. 1996; 78: 517–524.
25. Odley A, Hahn HS, Lynch RA, Marreez Y, Osinska H, Robbins J, Dorn GW 2nd. Regulation of cardiac contractility by Rab4-modulated ß2-adrenergic receptor recycling. Proc Natl Acad Sci U S A. 2004; 101: 7082–7087.
26. Patrucco E, Notte A, Barberis L, Selvetella G, Maffei A, Brancaccio M, Marengo S, Russo G, Azzolino O, Rybalkin SD, Silengo L, Altruda F, Wetzker R, Wymann MP, Lembo G, Hirsch E. PI3K modulates the cardiac response to chronic pressure overload by distinct kinase-dependent and -independent effects. Cell. 2004; 118: 375–387.[CrossRef][Medline]
[Order article via Infotrieve]
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