HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/3/523 02-0660fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by RAU, T. Articles by ESCHENHAGEN, T. Search for Related Content PubMed PubMed Citation Articles by RAU, T. Articles by ESCHENHAGEN, T.

Overexpression of wild-type Gi-2 suppresses ß-adrenergic signaling in cardiac myocytes¹

THOMAS RAU, MONIKA NOSE^*, UTE REMMERS^*, JOACHIM WEIL^*, ASTRID WEISSMÜLLER^*, KERRY DAVIA, SIAN HARDING, KARSTEN PEPPEL, WALTER J. KOCH and THOMAS ESCHENHAGEN²

Institute of Pharmacology and Toxicology, Friedrich Alexander University Erlangen, Germany;
^* Institute of Pharmacology and Toxicology, University Hospital, Hamburg, Germany;
Imperial College School of Medicine, NHLI, London, UK; and
Duke University School of Medicine, Durham, North Carolina, USA

²Correspondence: Institute for Pharmacology and Toxicology, Friedrich Alexander Universität Erlangen, Fahrstr. 17, 91054 Erlangen, Germany. E-mail: eschenhagen{at}pharmakologie.uni-erlangen.de

SPECIFIC AIMS

Downregulation of ß-adrenergic receptors and increased activity of the ß-adrenergic receptor kinase are established as factors involved in desensitization of ß-adrenergic signaling in human heart failure, whereas the role of overexpression of the inhibitory G-protein Gi-2 remains controversial. This study determined whether overexpression of wild-type Gi-2 impairs ß-adrenergic stimulation of adenylyl cyclase (AC) activity, cAMP formation, and cellular shortening in isolated rat myocytes.

PRINCIPAL FINDINGS

1. Gi-2 overexpression is virus dose dependent
Overexpression of rat wild-type Gi-2 in neonatal rat cardiac myocytes (NRCM) was achieved with an adenovirus encoding Gi-2 (Ad5Gi-2). Gi-2 levels increased by 20% to 600% depending on the multiplicity of infection (MOI 0.1–16 biologically active virus particles/cell; Fig. 1 A), whereas control virus (Ad5LacZ; ß-galactosidase; MOI 10) had no effect.

View larger version (44K): [in this window] [in a new window]   Figure 1. Virus dose-dependent Gi-2 overexpression: protein levels and effects on adenylyl cyclase activity. NRCM were infected with Ad5Gi-2 at increasing multiplicity of infection (MOI) for 48 h. 24 h before cell harvest, cells were treated with PTX or left untreated. A) Membrane proteins were subjected to in vitro 32P-ADP ribosylation and immunoblotted for the detection of Gi-2. In PTX-treated cells, no G-proteins were radioactively labeled in vitro. B) The same membranes were subjected to measurement of AC activity in the presence of 100 µM GTP or 100 µM GTP + 1 µM isoprenaline. Cells either were not treated with PTX or () or treated with PTX during the last 24 h before cell harvest (). Values are % of basal AC of uninfected controls. The effect of MOI on the attenuation of AC was highly significant for the stimuli tested (P<0.01, factorial ANOVA, n=6–9). *P < 0.05 vs. the respective uninfected control.

Gi-2-Overexpression is paralleled by attenuation of adenylyl cyclase activity
The effect of Gi-2 overexpression on AC activity was determined in membrane preparations of NRCM infected at increasing MOI for 48 h. AC activity was assessed under basal conditions and stimulation with GTP (100 µM) or GTP + isoprenaline (1 µM). Basal AC was virtually unaffected by Gi-2 overexpression. In contrast, the increase in Gi-2 was paralleled by a significant suppression of GTP- and isoprenaline/GTP-stimulated AC (P<0.001; Fig. 1B ). The effect of Ad5Gi-2 started at MOI 0.3, reached significance at MOI 1, and appeared to saturate at MOI 10–16 (Fig. 1B ). At MOI 1, GTP and isoprenaline/GTP-stimulated AC activity was reduced by 37% and 39%, respectively compared with uninfected controls (n=9, P<0.003). Ad5LacZ was without effect on stimulated AC at MOI of 10.

2. Pertussis toxin attenuates Gi-2 effects on adenylyl cyclase activity
PTX treatment (24 h before cell harvest) was used to assess specificity and reversibility of Gi-2 overexpression. Ribosylation was complete as demonstrated by subsequent in vitro PTX catalyzed with ³²P-ADP ribosylation (Fig. 1A ). PTX treatment completely offset the inhibitory effect of Gi-2 overexpression on GTP- and isoprenaline/GTP-stimulated AC up to MOI 1. At higher MOI, PTX only partially antagonized the Gi-2-mediated attenuation of AC (Fig. 1B ).

3. Gi-2 overexpression attenuates ß-adrenergic stimulation of cell shortening
Infection of adult rat ventricular myocytes (ARVM) with Ad5Gi-2 (MOI of 10³) increased Gi-2 to levels seen in NRCM at MOI 3. Ad5Gi-2 did not affect shortening or relaxation parameters at basal or maximum Ca²⁺ levels. However, the isoprenaline concentration-response curve was depressed and shifted to the right. The EC₅₀ increased from 4.2 ± 2.0 nM (uninfected myocytes) to 28.2 ± 1.3 nM (Ad5Gi-2; P=0.01). Maximal shortening amplitude was reduced from 11.4 ± 0.7% (control) to 8.3 ± 0.7% (Ad5Gi-2; P<0.01).

4. Gi-2 overexpression lowers cAMP levels in intact myocytes
An additional bicistronic adenovirus encoding wild-type Gi-2 and green fluorescent protein (GFP) under separate CMV promoters (Ad5Gi-2/GFP) was constructed to monitor transfection homogeneity. A GFP-only virus served as control (Ad5GFP). Infection of NRCM with Ad5Gi-2/GFP attenuated isoprenaline-stimulated cAMP accumulation by 55–95% in a virus dose-dependent manner (MOI 0.5–4, 48 h, P<0.001). Ad5GFP (MOI 4) had no effect. Basal cAMP levels were unchanged by Ad5Gi-2/GFP or Ad5GFP. Infection of NRCM with Ad5Gi-2/GFP or Ad5GFP (MOI 4, 48 h) did not affect the expression of Gs and Gß or the density of ß-adrenergic receptors.

5. Gi-2 overexpression targeted to 100% above control impairs isoprenaline-stimulated adenylyl cyclase activity and cAMP formation
An inherent problem of the approach to adjust Gi-2 overexpression levels by varying the virus dose is that, at low MOI, transfection efficiency is < 1, yielding inhomogeneous expression levels ranging from marked overexpression in positive cells to no overexpression in uninfected cells. Hence, by the approach used in the previous set of experiments (varying MOI, transfection for 48 h), it was not possible to obtain homogeneous levels of Gi-2 overexpression similar to those observed in human heart failure (i.e., by 50–100%). To solve this problem, a time course experiment was carried out using Ad5Gi-2/GFP at MOI 1.4. Twelve hours after infection, all cells were homogeneously transfected and Gi-2 levels were 93 ± 6% (Fig. 2 A; n=6) above control. Under this condition, GTP- and isoprenaline/GTP-stimulated Mn²⁺-normalized AC activity in broken membranes was reduced by 7 ± 3% and 17 ± 3%, respectively (Fig. 2B ; n=7; P<0.02), and isoprenaline-stimulated cAMP accumulation in NRCM was reduced by 40% compared with Ad5GFP-infected controls (Fig. 2C ; n=4; P<0.01). Experiments with specific receptor antagonists revealed that overexpression of Gi-2 attenuated ß1- and ß2-adrenergic stimulation of intracellular cAMP to the same relative extent (Fig. 2C ). The inverse antagonist atropine (1 µM) partially antagonized the Gi-2 effects.

View larger version (32K): [in this window] [in a new window]   Figure 2. Effect of moderate Gi-2 overexpression on cAMP levels in neonatal rat cardiac myocytes. A) Infection of NRCM with Ad5Gi-2/GFP at MOI 1.4 and cell harvesting 12 h after infection yielded a mean level of Gi-2 of 93% above control.. B) The same membranes were assayed for AC. Data were normalized for the respective Mn2+-stimulated AC activity. In membranes from Ad5GFP/Gi-2-infected NRCM, GTP- and isoprenaline/GTP-stimulated AC activity was 7% and 17% lower, respectively, than in membranes from Ad5GFP-infected controls (P<0.02). C) Infection of NRCM under identical conditions was used to determine the effect on isoprenaline-stimulated cAMP levels of myocytes. Isoprenaline (1 µM) was used in the presence or absence of the ß2- or ß1-selective antagonists ICI 118,551 (100 nM) or CGP 20712A (300 nM) or the inverse M-cholinoceptor agonist atropine (1 µM). In Ad5Gi-2/GFP-infected NRCM, cAMP levels were reduced by 40% compared to controls infected with Ad5GFP at the same MOI (n=4; P<0.01). ß1- and ß2-dependent cAMP formation was reduced to the same relative extent. The inverse M-cholinoceptor antagonist atropine partially antagonized the Gi-2 effect (P<0.05).

CONCLUSIONS AND SIGNIFICANCE

Desensitization of ß-adrenergic signaling is a hallmark of all forms of heart failure. It has been mainly attributed to down-regulation of ß1-adrenergic receptors and uncoupling of the receptors by increased activity of ß-adrenergic receptor kinases. In contrast, the role of the 50–100% increase of Gi-2 in failing hearts is controversial. Two reasons have been advanced against a substantial role of increased Gi-2. First, in heterologous transfection experiments only a mutant, constitutively active form of Gi-2 suppressed AC. Second, in view of the marked preponderance of inhibitory G-proteins over adrenergic receptors, Gs and AC, the moderate increase of Gi-2 observed in human heart failure has been considered by many to be of little relevance. However, some aspects of the perturbation of the AC pathways in heart failure are not explained by diminished ß1-adrenoreceptor density and coupling. 1) Desensitization involves not only the response to ß1- and ß2-adrenergic agonists, but also that to histamine and GTP (activating Gi and Gs). Since activation of AC by NaF (specific for Gs activation) and Mn²⁺ (G-protein independent) does not differ between failing and nonfailing human myocardium, the heterologous character of AC desensitization always pointed to Gi. 2) The impairment of contractile function and GTP-stimulated AC in myocytes from failing human hearts can be overcome by PTX treatment. 3) Levels of Gi-2 overexpression parallel the severity of heart failure and the extent to which responses to ß-adrenergic stimulation are blunted in vivo and in vitro. Thus, there is indirect evidence supporting a contribution of Gi-2 overexpression to the desensitization of ß-adrenergic signaling, but straight experimental evidence has been lacking.

Our results demonstrate that Gi-2 overexpression reduces isoprenaline-stimulated AC in membrane preparations and cAMP formation in intact NRCM. Moreover, isoprenaline-stimulated cell shortening of ARVM was blunted. The effect of Ad5-mediated overexpression of Gi-2 was surprisingly marked, dose-dependent, reversible by PTX, and occurred at a degree of Gi-2 overexpression resembling that in human heart failure.

Several reasons may explain the discrepancy between our results and previously published results demonstrating an impairment of cAMP signaling by mutant Gi-2 only. For example, the cellular background and gene transfer protocol were different. We used rat cardiac myocytes with the inherent complex signaling machinery of these cells, which may be lacking in NIH 3T3 fibroblasts. The adenovirus-based approach here allows efficient, virtually complete transfection of cells with rapidity, whereas the stable transfection used in the former study is prone to cellular adaptation and may obscure results. We took great care to adjust the levels of overexpression by varying both the virus dose and time of cell harvest to allow the construction of gene-dose effect curves in a homogeneously transfected cell population.

How can the effect of increased Gi-2 on AC be explained? Increases in Gi-2 could amplify signals from receptors with constitutive activity. Our results with atropine, an inverse agonist at muscarinic receptors that partially antagonized the consequences of Gi-2 overexpression, argue for this hypothesis. Support also comes from the reversal of Gi-2 effect by PTX, a toxin that interrupts interaction between receptors and Gi proteins without inactivating the G protein subunit itself. Yet the small effect of atropine and the fact that PTX reversed the Gi-2 effect only up to moderate levels may indicate that wild-type Gi-2 in itself has some intrinsic activity, i.e., spontaneously releases GDP in a PTX-insensitive manner. This could explain the almost complete abolition of AC and cAMP stimulation at high expression levels. Further, "nonspecific" effects at high expression levels such as binding of ß subunits cannot be excluded.

Collectively, the data suggest that 1) the Gi-2 system exhibits tonic inhibition of stimulated AC in cardiac myocytes, 2) Gi-2-mediated inhibition is concentration dependent and occurs at Gi-2 levels seen in heart failure, and 3) Gi-2-mediated inhibition affects ß1- and ß2-adrenergic stimulation of AC to a similar extent. The data argue for an important, independent role of the Gi-2 increase in heart failure that explains some characteristics of heart failure not attributable to down-regulation of ß-adrenergic receptors and their uncoupling from Gs.

View larger version (47K): [in this window] [in a new window]   Figure 3. Schematic diagram. Graphical depiction of the different molecular alterations implicated in the desensitization of adrenergic AC signaling in heart failure. Diminished receptor density and increased uncoupling of Gs-proteins act in concert with the negative input to AC generated by increased expression of Gi-2. Activation of Gi-2 may be partially mediated by empty M2-cholinoceptors.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0660fje; to cite this article, use FASEB J. (January 21, 2003) 10.1096/fj.02-0660fje

This article has been cited by other articles:

				M. Zhu, A. A. Gach, G. Liu, X. Xu, C. C. Lim, J. X. Zhang, L. Mao, K. Chuprun, W. J. Koch, R. Liao, et al. Enhanced calcium cycling and contractile function in transgenic hearts expressing constitutively active G{alpha}o* protein Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1335 - H1347. [Abstract] [Full Text] [PDF]

				H.-J. Hippe, M. Luedde, S. Lutz, H. Koehler, T. Eschenhagen, N. Frey, H. A. Katus, T. Wieland, and F. Niroomand Regulation of Cardiac cAMP Synthesis and Contractility by Nucleoside Diphosphate Kinase B/G Protein {beta}{gamma} Dimer Complexes Circ. Res., April 27, 2007; 100(8): 1191 - 1199. [Abstract] [Full Text] [PDF]

				D. S. Fernandez-Twinn, S. Ekizoglou, A. Wayman, C. J. Petry, and S. E. Ozanne Maternal low-protein diet programs cardiac beta-adrenergic response and signaling in 3-mo-old male offspring Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2006; 291(2): R429 - R436. [Abstract] [Full Text] [PDF]

				M. D. Richardson, J. D. Kilts, and M. M. Kwatra Increased Expression of Gi-Coupled Muscarinic Acetylcholine Receptor and Gi in Atrium of Elderly Diabetic Subjects Diabetes, September 1, 2004; 53(9): 2392 - 2396. [Abstract] [Full Text] [PDF]

				A. El-Armouche, O. Zolk, T. Rau, and T. Eschenhagen Inhibitory G-proteins and their role in desensitization of the adenylyl cyclase pathway in heart failure Cardiovasc Res, December 1, 2003; 60(3): 478 - 487. [Abstract] [Full Text] [PDF]

				M. J. Lohse, S. Engelhardt, and T. Eschenhagen What Is the Role of {beta}-Adrenergic Signaling in Heart Failure? Circ. Res., November 14, 2003; 93(10): 896 - 906. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/3/523 02-0660fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by RAU, T. Articles by ESCHENHAGEN, T. Search for Related Content PubMed PubMed Citation Articles by RAU, T. Articles by ESCHENHAGEN, T.