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Circulation Research. 2007;100:1191-1199
Published online before print March 15, 2007, doi: 10.1161/01.RES.0000264058.28808.cc
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(Circulation Research. 2007;100:1191.)
© 2007 American Heart Association, Inc.

Cellular Biology

Regulation of Cardiac cAMP Synthesis and Contractility by Nucleoside Diphosphate Kinase B/G Protein ß{gamma} Dimer Complexes

Hans-Joerg Hippe, Mark Luedde, Susanne Lutz, Henrike Koehler, Thomas Eschenhagen, Norbert Frey, Hugo A. Katus, Thomas Wieland*, Feraydoon Niroomand*

From the Innere Medizin III – Kardiologie (H.-J.H., M.L., H.K., N.F., H.A.K., F.N.), Universität Heidelberg, Heidelberg, Germany, §Institut für Experimentelle und Klinische Pharmakologie und Toxikologie (S.L., T.W.), Universität Heidelberg, Mannheim, Germany, Institut für Experimentelle und Klinische Pharmakologie und Toxikologie (T.E.), Universität Hamburg, Hamburg, Germany.

Correspondence to Hans-Joerg Hippe, M.D., Innere Medizin III – Kardiologie, Universität Heidelberg, INF 410, D-69120 Heidelberg, Germany. E-mail hans-joerg.hippe{at}med.uni-heidelberg.de


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
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Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein {alpha} subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein ß{gamma}-dimers (Gß{gamma}). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a Gß1{gamma}2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the ß1 subunit (Gß1H266L{gamma}2). Recombinantly expressed Gß1H266L{gamma}2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate Gß phosphorylation. Compared with wild-type Gß1{gamma}2 (Gß1WT{gamma}2), overexpression of Gß1H266L{gamma}2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/Gß{gamma} complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing Gß1H266L{gamma}2, the basal contractility was suppressed by {approx}50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the ß-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in Gß1H266L{gamma}2 than in Gß1WT{gamma}2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by Gß1H266L{gamma}2. We conclude that the receptor-independent activation of G proteins via NDPK B/Gß{gamma} complexes requires the intermediate phosphorylation of G protein ß subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for Gß and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.


Key Words: G proteins • NDPK • cardiomyocytes • cAMP • contractility


*    Introduction
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 up arrowAbstract
 *Introduction
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Heterotrimeric GTP-binding proteins (G proteins) are important mediators in many signal transduction pathways.1 In cardiomyocytes, activation of Gs and Gi proteins via G protein-coupled receptors (GPCR) regulates intracellular cAMP levels, which control myocardial contractility by activation of protein kinase A (PKA) pathway and alteration in Ca2+ transients.2,3 It is generally accepted that heterotrimeric G proteins are activated by a GPCR-induced GDP/GTP exchange at the G{alpha} subunit, followed by regulation of specific effector molecules, such as adenylyl cyclase (AC), phospholipase Cß or ion channels.1,4

In addition, GTP formation by nucleoside diphosphate kinases (NDPKs) contributes to the activation of G proteins, a concept first described by Kimura and coworkers.5 Recent data suggest that a NDPK-dependent phosphate relay acts as alternative pathway to the GPCR-induced GDP/GTP exchange.6,7 NDPKs, encoded by nm23 genes, represent an evolutionarily and structurally conserved family of multifunctional proteins that have been implicated in various cellular processes, including tumor metastasis,8,9 apoptosis,10 endocytosis,11 and gene regulation.12 In eukaryotic cells, the major isoforms NDPK A, B, and C (17 to 21 kDa) are characterized by their enzymatic activity, ie, generation of nucleoside triphosphates from the respective diphosphates through a high-energy His-118 intermediate.13 Only a small fraction of NDPK is associated with the plasma membrane. However, it has been reported that the NDPK content at the plasma membrane is increased by 300% in hearts from patients with congestive heart failure14 and in hearts from rats with isoproterenol-induced hypertrophy.15

We have recently shown that Gs proteins can be activated by a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and the ß{gamma} subunit of G proteins.6,7 The complex formation of NDPK B with Gß{gamma} dimers was detected in different tissues. Within this complex, NDPK B apparently phosphorylates Gß at His-266. The phosphate is of high energy and can be transferred onto GDP. The formed GTP then subsequently activates Gs and Gi proteins.16,17 In accordance with this mechanism stable overexpression of NDPK B, but not of its catalytically inactive mutant NDPK B-H118N or NDPK A, largely enhanced G{alpha}s-dependent cAMP formation in living cells.7

To analyze the contribution of the NDPK B/Gß{gamma} complex to G protein activation in cardiomyocytes, we generated a mutant of the G protein ß1 subunit, in which the intermediately phosphorylated His-266 is substituted by leucine (Gß1H266L). We show that adenoviral overexpression of Gß1H266L{gamma}2-dimers as well as siRNA-induced depletion of NDPK B reduced receptor-independent G{alpha}s activation in cardiomyocytes. Thus, the NDPK B/Gß{gamma}-mediated phosphate transfer is apparently involved in the regulation of receptor-independent cAMP synthesis and contractility in cardiomyocytes.


*    Materials and Methods
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 *Materials and Methods
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 down arrowDiscussion
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Generation of Recombinant Adenoviruses
Human cDNA of Gß1 and G{gamma}2 were obtained from UMR cDNA Resource Center, University of Missouri-Rolla. Site directed mutagenesis (QuikChange, Stratagene) was performed to introduce leucine for histidine at position 266 of Gß1 according to the manufactures protocol. cDNA encoding Gß1 was subcloned into the pShuttle-IRES-hrGFP-1 vector (Stratagene). Gß1 in conjunction with the internal ribosome entry site (IRES) was isolated from pShuttle and subcloned into the pAdTrack-CMV vector.18 G{gamma}2 was inserted into this construct downstream the IRES to obtain bicistronic mRNA. Recombinant adenoviruses were generated by homologous recombination with the adenoviral backbone vector pAd-Easy-1.18 Adenoviruses encoding for GFP alone and the long form of rat G{alpha}s has been described.7

Isolation and Infection of Rat Cardiomyocytes
Culture of immortalized neonatal rat cardiomyocytes (H10 cells) and generation of stably transfected cell clones (NDPK+ cells) has been described.7 Neonatal rat cardiomyocytes (NRCM) were isolated as described19 and were infected with the indicated multiplicity of infection (MOI) under serum-free conditions 48 hours after plating and subsequently cultured supplemented with 2% FCS for 48 hours. Adult rat cardiomyocytes (ARCM) were isolated from Sprague-Dawley rats using a collagenase digestion method19 and were infected 1 hour after plating and further cultured for 48 hours in a HEPES-modified medium 199 (M199, Sigma S7528, supplemented with 5 mmol/L taurine, 5 mmol/L carnitine, 5 mmol/L creatine, 5 mmol/L N-mercaptoproprionyl glycine, 0.1 µmol/L insulin, 10,000 U/mL penicillin and 10 mg/mL streptomycin, pH 7.25).

NDPK Depletion in NRCM
Knockdown was performed by a sequential, double transfection with siRNA duplexes (100 nmol/L) as described20 using Lipofectamine2000 reagent (Invitrogen). siRNAs were directed against the following target sequences10,20: NDPK A#1: GGATTCCGCCTGGTTGGTT, NDPK B#1: GGGGTTCCGCCTGGTGGCC, NDPK B#2: AACTGATTGACTATAAGTCTT. Control siRNA (si-Control) was a scrambled siRNA (Ambion).

Measurement of Contractile Parameters
Contractile parameters in ARCM were obtained 48 hours after adenoviral gene transfer by video edge detection, as described previously21 at 1.8 mmol/L [Ca2+]e and at 0.5 Hz field stimulation.

Phosphorylation of Gß and NDPK in Cell Membranes
Preparation of cell membranes and phosphorylation of Gß was performed exactly as described previously.22

Measurement of cAMP Levels
Generation of cAMP was assayed in serum-free medium containing 1 mmol/L 3-isobutyl-1-methylxanthine (IBMX) in the presence of propranolol or isoproterenol (ISO) with 100 µmol/L ascorbic acid as indicated for 20 minutes as previously described22 using a cAMP immunoassay (R&D Systems).

Western Blotting
Western blotting was performed according to standard procedures.22 Immunodetection was performed by using specific primary antibodies against G{alpha}s (Gramsch), Gßcom, Gß1, G{gamma}2, NDPK (Santa Cruz), G{alpha}i2 (PerkinElmer Life Science), calsequestrin (Swant), pan-Cadherin (Abcam), Ser16-phospho-phospholamban (Upstate) and ß-actin (Sigma). Specific NDPK B antibody was a kind gift of Dr Ioan Lascu, Bordeaux. Detection was performed with suitable secondary antibodies and an enhanced chemiluminescence (ECL) reagent (Pierce).

Coimmunoprecipitation
Cells were lysed in immunoprecipitation buffer (10 mmol/L Tris-HCl, pH 7.4, 0.1% Triton X-100, 150 mmol/L NaCl, 1 mmol/L PMSF and protease inhibitors) 48 hour after infection. Coimmunoprecipitation was performed as described7 using anti-G{alpha}s antibody and the precipitate was immunoblotted for G{alpha}s and Gß.

Statistical Analysis
All results were expressed as mean±SEM. We performed statistical analysis using one-way ANOVA with Bonferroni post hoc test or t-tests as appropriate. P<0.05 was considered statistically significant.


*    Results
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 *Results
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Integration of Phosphodeficient Gß1H266L{gamma}2 Dimers Into Heterotrimeric Gs Proteins
To analyze the importance of the NDPK B/Gß{gamma} complex for G protein activation, we constructed a mutant of the G protein ß1 subunit, in which the intermediately phosphorylated His-266 residue was changed to a leucine (Gß1H266L). We generated biscistronic adenoviral vectors encoding wild-type Gß1 (Gß1WT{gamma}2) or H266L-mutated Gß1 (Gß1H266L{gamma}2) in conjunction with G{gamma}2, to allow for formation of Gß{gamma}-dimers in the target cell.

Gß1WT{gamma}2 or Gß1H266L{gamma}2 dimers were adenovirally overexpressed in H10 cells and NRCM. An adenovirus encoding GFP alone (AdGFP) served as additional control. Overexpressed Gß1WT{gamma}2 and Gß1H266L{gamma}2 were localized in the membrane fraction of infected myocytes (Figure 1A), which is in accordance with the known localization of correctly formed and folded Gß{gamma} dimers.23 The content of the membrane in G{alpha}s and G{alpha}i was slightly increased, most likely because of enhanced heterotrimer formation.


Figure 1
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  		Figure 1. Overexpression of Gß{gamma} dimers and NDPK-mediated phosphorylation of G protein ß subunits in cardiomyocytes. A, Cell membranes prepared 48 hour after infection with adenoviruses encoding GFP, Gß1WT{gamma}2 or Gß1H266L{gamma}2 were subjected to immunoblot analysis with specific antibodies for Gß1, G{gamma}2, Gs and Gi2. Similar data were obtained in H10 cells and NRCM. B, Integration of overexpressed Gß1{gamma}2-dimers, wild-type or H266L-mutant, into heterotrimeric Gs proteins was confirmed by immunoprecipitation (IP) with an anti-G{alpha}s antibody from NRCM lysates infected as indicated. The precipitates were probed for Gß with a Gß1- and a more sensitive Gßcom-specific antibody. C, Cell membranes were phosphorylated with [{gamma}-32P]GTP for 1 and 3 minutes at 30°C. A representative autoradiography after SDS-Page of three independent experiments is shown. Phosphorylated Gß and autophosphorylated NDPK are indicated.

To verify the integration of overexpressed wild-type and mutated Gß1{gamma}2-dimers into heterotrimeric G proteins coimmunoprecipitations were performed in NRCM infected with AdGß1{gamma}2. As shown in Figure 1B, an anti-G{alpha}s-antibody coprecipitated similar amounts of Gß1WT and Gß1H266L bound to endogenous G{alpha}s. When G{alpha}s-expression was increased by AdG{alpha}s increasing amounts of overexpressed Gß1WT and Gß1H266L were coprecipitated. These findings indicate a competition of the overexpressed Gß1{gamma}2-dimers with endogenous Gß{gamma} for heterotrimer formation with G{alpha}s.

To test whether Gß1H266L{gamma}2 is a substrate for the NDPK B phosphorylation of Gß, cell membranes were phosphorylated with [{gamma}-32P]GTP. As shown in the autoradiography (Figure 1C), overexpression of Gß1WT{gamma}2 increased the intermediate Gß-phosphorylation (2.1±0.2 -fold, normalized to GFP control, n=3). In contrast, Gß-phosphorylation did not increase above endogenous level when Gß1H266L{gamma}2 was overexpressed (Figure 1C). These results confirm that residue His-266 is the phosphorylated amino acid in Gß1 and demonstrate that H266L-mutated Gß1 cannot be phosphorylated by NDPK B.

In summary, these data indicate a similar efficient incorporation of the phosphodeficient Gß1H266L into heterotrimeric Gs as Gß1WT, providing an efficient tool to study the effect of this point mutation on G protein activity.

NDPK B-Dependent Suppression of Basal, Receptor-Independent cAMP Synthesis by Overexpression of Gß1H266L{gamma}2
Previous findings in H10 cells7 suggest that NDPK B/Gß{gamma}-mediated phosphate transfer is a mechanism for receptor-independent G protein activation. Here we studied the effect of a His to Leu replacement at position 266 in Gß1 on NDPK B/Gß{gamma}-mediated Gs activation, using cAMP as reporter. Experiments were performed in H10-WT cells and H10 cells with a 3-fold overexpression of NDPK B (NDPK+ cells7). cAMP production was determined in the presence of the ß-adrenoceptor (ßAR) inverse agonist propranolol to block spontaneous or constitutive ßAR activity. Infection of H10 cells with increasing doses of AdG{alpha}s combined with either AdGß1WT{gamma}2 or AdGß1H266L{gamma}2 resulted in similar amounts of protein expression (Figure 2A). Overexpression of the Gß1{gamma}2 variants did not alter of NDPK B expression (Figure 2A) nor the total NDPK activity in these cells (GFP: 93.7±4.3, Gß1WT{gamma}2: 90.5±3.6, Gß1H266L{gamma}2: 91.7±4.3 nmol/min*mg). In H10-WT cells, increasing amounts of G{alpha}s were associated with a linear increase cAMP accumulation. However, coexpression of Gß1H266L{gamma}2 reduced this effect by {approx}30% compared with Gß1WT{gamma}2 at each level of G{alpha}s (Figure 2B). In NDPK+ cells, the elevated NDPK B content increased the G{alpha}s-mediated cAMP synthesis7 and enhanced the inhibitory effect of Gß1H266L{gamma}2 (Figure 2C). At the highest G{alpha}s expression (MOI 100), the cAMP content in Gß1H266L{gamma}2-infected NDPK+ cells amounted to 45.0±3.7% (n=4, P<0.001) of that seen in Gß1WT{gamma}2-infected cells. Thus, the observed difference between Gß1WT{gamma}2 and Gß1H266L{gamma}2 overexpressing cells in basal cAMP synthesis apparently depends on both the level of G{alpha}s and NDPK B.


Figure 2
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  		Figure 2. Expression of Gß1H266L{gamma}2 reduces cAMP production in H10 cells in a NDPK B and G{alpha}s dependent manner. A, Immunoblot analysis of lysates from H10-WT (left panel), H10-WT and NDPK+ cells (right panel), infected at MOI 100, if not otherwise indicated, and probed with specific antibodies against NDPK B, Gß1 and G{alpha}s. B and C, Basal cAMP content was determined in H10-WT cells (B) and in NDPK+ cells 3-fold overexpressing NDPK B (C) in the presence of 1 mmol/L IBMX and 1 µmol/L propranolol 48 hour after adenoviral infection with AdGß1WT{gamma}2 or AdGß1H266L{gamma}2 (MOI 100) combined with AdG{alpha}s at the indicated MOI. The inhibition of the cAMP content in Gß1H266L{gamma}2 expressing cells related to Gß1WT{gamma}2 expressing cells is shown at the right panels in B and C. Data are means±SEM, n=4; *P<0.05; **P<0.01; §P<0.001 vs Gß1WT{gamma}2; #P<0.05; ##P<0.01 between NDPK+ and H10-WT cells.

H10 cells are derived from NRCM, but are altered by the immortalization. Therefore, we performed a similar coexpression of G{alpha}s with either Gß1WT{gamma}2 or Gß1H266L{gamma}2 in freshly isolated NRCM and determined the cAMP accumulation in the presence of propranolol. Note that NRCM are more sensitive to adenoviral infection than H10 cells. Thus, for similar effective adenoviral infections a 10-fold reduced MOI was sufficient (Figure 3A). Overexpression of Gß1WT{gamma}2 did not alter basal cAMP compared with GFP controls (GFP: 60.7±7 pmol/mg; Gß1WT{gamma}2: 57.7±10 pmol/mg). In NRCM, we observed two effects of Gß{gamma} overexpression on cAMP formation: (1) when compared with the expression of G{alpha}s alone (Figure 3B), coexpression of G{alpha}s and Gß{gamma} dimers resulted in a strong reduction (89±3%) in basal cAMP synthesis, which is because of the well known formation of inactive G{alpha}sß{gamma} heterotrimers (see Figure 1B) in the absence of a stimulated receptor24; and (2) beyond this expected effect, overexpression of Gß1H266L{gamma}2 induced a further significant suppression of the receptor-independent cAMP synthesis. Similarly to NDPK+ cells, cAMP levels of Gß1H266L{gamma}2 myocytes were reduced by 30% to 50% compared with Gß1WT{gamma}2 myocytes in a G{alpha}s level dependent manner (Figure 3C).


Figure 3
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  		Figure 3. Suppression of basal cAMP production in NRCM by expression of Gß1H266L{gamma}2. NRCM were coinfected with AdG{alpha}s at the indicated MOI and AdGFP or AdGß1{gamma}2, either WT or H266L-mutant, at MOI 10. A, Representative immunoblots of the expression of Gß1 and G{alpha}s 48 hour after infection. B and C, Comparison of basal cAMP production in the presence of 1 mmol/L IBMX and 1 µmol/L propranolol expressing GFP or Gß1WT{gamma}2 (B) and Gß1WT{gamma}2 or Gß1H266L{gamma}2 (C). Data are means ± SEM, n=4; *P<0.05; **P<0.01.

Specific Depletion of NDPK B Decreases Basal cAMP Synthesis in NRCM
To interfere with the phosphate transfer reactions by NDPK B/Gß{gamma} complexes using a different approach, we performed siRNA-mediated knockdown of NDPK isoforms in NRCM. A sequential, double transfection with siRNA duplexes targeting NDPK A (si-NDPK A) and B (si-NDPK B#1, B#2) specifically reduced the expression of the targeted NDPK isoform by approximately 70% (Figure 4A). Depletion of NDPK B, but not of NDPK A, resulted in significantly diminished cAMP levels (si-NDPK B#2: 47.8±2.6% of control, n=3, P<0.01) which correlated with the reduction in NDPK B expression (Figure 4B). This effect is likely because of the attenuation of NDPK B/Gß{gamma} complex formation by NDPK B depletion.


Figure 4
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  		Figure 4. NDPK B depletion decreases basal cAMP production in NRCM. A, Representative immunoblots of total NDPK and specific NDPK B expression in NRCM 72 hour after transfection with scrambled siRNA (si-Control) or specific siRNA (si-) against NDPK A or B (#1, #2) as indicated. Expression levels of ß-actin and pan-Cadherin are shown as loading controls. B, Basal cAMP contents were measured in NRCM 72 hour after transfection with the indicated siRNA in the presence of 1 mmol/L IBMX and 1 µmol/L propranolol. Data are given as percentage of cAMP content in si-Control transfected cells (56.4±3.2 pmol/mg). Data are means±SEM, n=4; *P<0.05; **P<0.01.

Suppression of Basal, Receptor-Independent Contractility of Cardiomyocytes by Overexpression of Gß1H266L{gamma}2
Changes in cAMP formation influence contractility via activation of PKA and subsequent modulation of Ca2+-transients by phosphorylation of proteins involved in Ca2+-handling, eg, phospholamban (PLB).25 We therefore studied the impact of impaired phosphorylation of Gß at His-266 on cAMP levels, PLB phosphorylation and contractility in ARCM. Efficient transduction with adenoviruses encoding GFP, Gß1WT{gamma}2 or Gß1H266L{gamma}2 was monitored by GFP expression driven by an independent CMV promoter (Figure 5A). The viability of the infected cells (65% to 70% after 48 hour, detected by trypan blue exclusion) was similar for all used adenoviruses. Immunoblots of whole cell lysates confirmed similar levels of overexpression of Gß1WT{gamma}2 or Gß1H266L{gamma}2 at the indicated MOI (Figure 5A). Concordant with H10 cells and NRCM, basal cAMP levels were significantly reduced in ARCM expressing Gß1H266L{gamma}2 by 35±2% (n=5, P<0.05) compared Gß1WT{gamma}2 (Figure 5B). Gß1H266L{gamma}2 overexpression also suppressed PLB phosphorylation by 40±9% (n=3, P<0.05) relative to control myocytes (Figure 5C). Single-myocyte contractility was analyzed using video edge detection in the presence of propranolol.19 Compared with AdGFP-infected cells (MOI 104), uninfected ARCM exhibited no significant differences in the contraction parameters analyzed either with or without 100 nmol/L propranolol (data not shown). Overexpression of Gß1WT{gamma}2-dimers slightly, but significantly increased myocyte contractility when compared with AdGFP-infected myocytes (fractional shortening: Gß1WT{gamma}2 versus GFP: 13.3±0.3% versus 10.7±0.3%, n=81 to 95, P<0.05) (Figure 5D). This might reflect a cAMP-independent effect of the well known Gß{gamma} interaction with cardiac ion channels or other Gß{gamma} regulated pathways. Overexpression of Gß1H266L{gamma}2, however, significantly reduced myocyte contractility compared with Gß1WT{gamma}2 and GFP overexpressing myocytes. At the highest expression level of Gß1{gamma}2 (MOI 104), basal fractional shortening in Gß1H266L{gamma}2 expressing myocytes (7.4±0.4%) was reduced to {approx}50% of that observed in Gß1WT{gamma}2 overexpressing controls (13.3±0.3%, n=81, P<0.01). Similar decreases in maximum rates of myocytes shortening (–dl/dt) and relengthening (+dl/dt) were found in Gß1H266L{gamma}2 myocytes (Figure 5D). As observed before (Figure 2 and 3Up) additional overexpression of G{alpha}s enhanced the difference between AdGß1H266L{gamma}2- and AdGß1WT{gamma}2-infected myocytes (supplemental Figure I in the online data supplement available at http://circres.ahajournals.org).


Figure 5
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  		Figure 5. Suppression of basal cAMP formation, PLB phosphorylation and contractility by expression of Gß1H266L{gamma}2 in ARCM. A, Representative transmission and GFP emission images of ARCM 48 hour after infection with AdGFP, AdGß1WT{gamma}2 or AdGß1H266L{gamma}2 at a MOI 104, respectively and representative immunoblots of whole cell lysates at the indicated MOI with specific antibodies for Gß1, G{alpha}s, NDPK B and calsequestrin (CSQ) are shown. Scale bar: 100 µm. B and C, Basal cAMP levels (B) and immunoblot analysis of Ser16-phosphorylated phospholamban monomers (P-PLB) and Gß1 (C) in the presence of 1 mmol/L IBMX and 100 nmol/L propranolol in ARCM (MOI 104). Calsequestrin (CSQ) served as loading control. Data are means±SEM, n=3; *P<0.05 vs Gß1WT{gamma}2; #P<0.05 vs GFP. D, Fractional shortening (% of cell length), rate of myocyte shortening (–dl/dt) and myocyte relengthening (+dl/dt) were measured in ARCM infected with AdGß1WT{gamma}2 or AdGß1H266L{gamma}2 at the indicated MOI in the presence of 100 nmol/L propranolol. Data are from 5 different myocyte preparations, n=50 to 80 cells per condition and are means±SEM. *P<0.05; **P<0.01 vs Gß1WT{gamma}2; #P<0.05 vs GFP.

Thus, an impaired phosphorylation of Gß1 at His-266 in cardiomyocytes leads to a substantial suppression of basal cAMP, PLB phosphorylation and receptor-independent contractility.

Impact of Gß1H266L{gamma}2 on cAMP Formation and Cardiomyocyte Contractility in the Presence of Isoproterenol
The activation of G proteins via ßARs plays a key role in the regulation of cardiac contractility.3 We therefore analyzed the induction of cAMP synthesis and contractility in response to the ßAR agonist isoproterenol (ISO) in H10 cells, NRCM, and ARCM overexpressing Gß1WT{gamma}2 or Gß1H266L{gamma}2 at a similar level. The total values of cAMP formation in the presence of ISO were significantly lower by {approx}30% in Gß1H266L{gamma}2 expressing cells compared with Gß1WT{gamma}2 (Figure 6A–C). Nevertheless, both Gß1WT{gamma}2 and Gß1H266L{gamma}2 expressing cells exhibited similar responses to ISO stimulation in either cell type, when related to the different basal cAMP levels (Figure 6A–C, insets, -fold stimulation).


Figure 6
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  		Figure 6. Effect of Gß1H266L{gamma}2 on isoproterenol-stimulated cAMP production and contractility. A–C, Isoproterenol (ISO)-stimulated cAMP production. H10 cells (A), NRCM (B) and ARCM (C) were infected with AdGß1WT{gamma}2 or AdGß1H266L{gamma}2 (MOI 100, 10, 104, respectively) and stimulated with ISO at the indicated concentrations. Total values in cAMP formation (n=3) as well as -fold stimulation (Insets) are shown. The expression of the respective Gß1 proteins was monitored by immunoblotting (Insets). Data are means±SEM, n=3 to 4; *P<0.05; **P<0.01. D, ARCM were infected with AdGFP, AdGß1WT{gamma}2 or AdGß1H266L{gamma}2 (MOI 104). Fractional shortening was measured in unstimulated cells (Basal) and in cells stimulated with 10 nmol/L ISO. {Delta}, basal contraction was subtracted from ISO-induced amplitudes. Data are from 6 different myocyte preparations and are means±SEM, n=48 to 61; *P<0.05 vs Gß1WT{gamma}2; #P<0.05 vs GFP.

Contraction amplitudes of ARCM increased up to 2-fold in response to ISO (Figure 6D). When compared with AdGFP- or AdGß1WT{gamma}2- infected myocytes the ISO-induced increase in contraction (Figure 6D, {Delta}, ISO-Basal) was virtually unchanged in Gß1H266L{gamma}2 expressing cells. In accordance to the cAMP data, the absolute contraction amplitudes in the presence of ISO were significantly attenuated in AdGß1H266L{gamma}2-infected myocytes, which likely reflects the reduced basal contractility in these cells (Figure 6D).

In addition to these functional data, we used a rather artificial in vitro system, ie, baculovirus-mediated expression of G{alpha}sß1H266L{gamma}2, G{alpha}sß1WT{gamma}2 and ßAR in Sf9 insect cells, to directly measure the ßAR-induced GDP/GTP exchange (supplemental Figure II in the online data supplement). We observed no difference in ßAR-induced G{alpha}sß1H266L{gamma}2 or G{alpha}sß1WT{gamma}2 activation in Sf9 cells.

Taken together, these findings indicate that an impaired phosphorylation of Gß1 at His-266 in cardiomyocytes does not compromise G protein activation by receptor-stimulated GDP/GTP exchange but significantly attenuates total G protein activity also in the presence of a receptor agonist.


*    Discussion
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 *Discussion
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According to the concept first described by Gilman4 heterotrimeric G proteins are transducers of extracellular signals from cell surface G protein-coupled receptors (GPCR) to intracellular effectors. We previously complemented this concept by showing that NDPK B is a receptor-independent activator of G proteins in living cells.7 Within a NDPK B/Gß{gamma} complex, the NDPK B apparently transfers a high energetic phosphate via intermediately phosphorylated G protein ß-subunits specifically onto GDP. This GTP formation induces the activation of the G protein. The intermediately phosphorylated amino acid was identified as His-266 in Gß.6 In the present study, we confirmed the identity of His-266 as the phosphorylated amino acid (see Figure 1C). The substitution of leucine for histidine 266 in Gß1 led to the formation of a Gß1{gamma}2-dimer, which in contrast to wild-type Gß1{gamma}2 could no longer be phosphorylated by NDPK B. We obtained several lines of evidence that the Gß1H266L{gamma}2-dimer forms regular heterotrimers with G{alpha}s and that the activation of such heterotrimers via the classical GPCR-induced GDP/GTP exchange is unaffected. First, Gß1WT{gamma}2 and Gß1H266L{gamma}2 could be coimmunoprecipitated with endogenous or overexpressed G{alpha}s to a similar extent. Second, although the total values in response to ISO were reduced by Gß1H266L{gamma}2, the ISO-induced stimulation in cAMP synthesis and contractility were comparable in myocytes expressing Gß1WT{gamma}2 or Gß1H266L{gamma}2. Particularly, when related to the different baseline levels in myocytes expressing Gß1WT{gamma}2 or Gß1H266L{gamma}2 virtually no alterations in the extent (x-fold) or concentration dependency of the stimulation by ISO occurred. Third, baculovirus-mediated expression of Gß1H266L{gamma}2 or Gß1WT{gamma}2 similarly supported the ßAR-induced activation of G{alpha}s in Sf9 insect cells. Unlike the mutation of NDPK B at His-118,26 which interferes with all processes requiring the catalytic activity of NDPK B, the H266L-mutant of Gß1 therefore provided a tool to selectively disrupt NDPK B-mediated G protein activation without apparently interfering with processes requiring NDPK B activity on the one hand, or GPCR-induced GDP/GTP exchange on the other.

Whereas receptor-induced control of cAMP synthesis and the resulting response in contractility have been studied in great detail,2,27 little is known about the control of basal, receptor-independent cAMP formation in and contractility of cardiomyocytes. As our previous data6,7,15 indicated a contribution of the NDPK B/Gß{gamma} complex to basal, receptor-independent activation of heterotrimeric G proteins, we adenovirally overexpressed Gß1H266L{gamma}2 and Gß1WT{gamma}2 in H10 cells, NRCM and ARCM to study the ability of the H266L-mutated Gß1 to interfere with the receptor-independent activation of G{alpha}s. To exclude influences of a possible constitutive activity of ßARs (for review see Ref. 28) assays were performed in the presence of the inverse ßAR agonist propranolol. Overexpression of Gß{gamma} in cells may induce several effects. In accordance with data in the literature29,30 we observed a slight increase in the Gs and Gi content in the membrane fraction (see Figure 1A). Moreover, free Gß{gamma}-dimers can induce a costimulation of Gß{gamma}-sensitive AC isoforms in the presence of activated G{alpha}s31 which would perturb cAMP formation and contractility measurement. Nevertheless, the major cardiac AC isoforms type V and VI32,33 are not costimulated by Gß{gamma}. Accordingly, no increase in cAMP was observed in H10 cells, NRCM and ARCM. However, compared with the expression of G{alpha}s alone, we observed a strong reduction in cAMP content when Gß{gamma} is coexpressed with G{alpha}s (see Figure 3B). This prominent decrease likely represents the well known formation of inactive heterotrimers (see also Figure 1B) in which the G{alpha} subunits are stabilized in their GDP-bound form by Gß{gamma} binding.24

As Gß1WT{gamma}2 and Gß1H266L{gamma}2 did not differ in their ability to form G{alpha}sß{gamma} heterotrimers (see Figure 1B), we conclude that the additional reduction in cAMP synthesis and the suppression of basal contractility detected when His-266 in Gß1 is replaced by leucine (see Figure 2, 3, and 5UpUp) reflects the lack of activation of G{alpha}s via NDPK B/Gß{gamma}-mediated phosphate transfer. This interpretation is further supported by the data showing that the decrease in cAMP and contractility is further enhanced by elevated expression of NDPK B (see Figure 2) and Gß1{gamma}2 dimers (see Figure 5), respectively. Apparently, Gß1H266L{gamma}2 is effectively competing with endogenous Gß{gamma} for complex formation with NDPK B and thus the impact of Gß1H266L{gamma}2 on cAMP synthesis is more pronounced in NDPK+ cells. Moreover, reducing the formation of NDPK B/Gß{gamma} complexes by siRNA-mediated depletion of NDPK B content (see Figure 4) suppressed the basal cAMP production in NRCM. These observations are in line with previous data obtained in H10 cells overexpressing NDPK B or its catalytic inactive H118N-mutant.7 In addition, increased expression of G{alpha}s enhanced the difference between Gß1H266L{gamma}2 and Gß1WT{gamma}2 overexpressing myocytes (see Figure 2, 3Up, and supplemental Figure I available at http://circres.ahajournals.org). Therefore, the data at large indicate that the contribution of the NDPK B/Gß{gamma}-mediated phosphotransfer to basal G{alpha}s activation is the stronger the more G{alpha}s/Gß{gamma}/NDPK B complexes are able to form. Cellular depletion of the complex partner NDPK B or incorporation of mutants defective in phosphotransfer (NDPK B-H118N7 or even more specific Gß1H266L) into such complexes blocks this activation pathway. Noteworthy, also effectors which might be activated directly by G{alpha}s and contribute to basal contractility in cardiomyocytes, eg, L-type Ca2+ channels,34 are potentially regulated by G{alpha}s/Gß{gamma}/NDPK B complexes.

If the formation of such G{alpha}ß{gamma}/NDPK B complexes plays a role in basal G protein activity, the question is raised, whether its contribution is regulated itself. In hearts from patients with severe congestive heart failure a 3-fold elevated content and activity of NDPK in plasma membranes was detected.14 This elevation was diminished in patients treated with a ßAR antagonist. Chronic treatment of rats with the ßAR agonist ISO, induced an increase in plasma membrane-bound NDPK,15 suggesting chronic ßAR activation as a mechanism for regulation of the NDPK membrane content. As Gi protein expression is also increased in heart failure,35 the higher NDPK and Gi content may lead to an increased formation of G{alpha}iß{gamma}/NDPK B complexes, which cause diminished basal cAMP formation and, thus, decreased contractility.35 In this context, it is noteworthy that NDPK B apparently acts as a (G protein-) histidine kinase in that complex. Thus, it might be of particular interest that the first identified eukaryotic phosphohistidine phosphatase (PHP)36 specifically dephosphorylates Gß, but not NDPK B, in reconstituted systems and in H10 cells membranes.37 Therefore, PHP might be a candidate for an endogenous regulator of basal NDPK-dependent G protein activation targeting the same specific step in the activation, which we artificially suppressed by mutating His-266 in Gß1. Although currently no data are available with regard to His-266 phosphorylation of Gß-subunits under pathophysiological conditions, NDPK is playing a role in human heart failure14 and rat heart hypertrophy.15 Therefore, the modulation of His-266 phosphorylation of Gß could be a potential novel target for therapeutical interventions in heart failure.

In conclusion, our data show that the basal, receptor-independent activation of cardiac Gs proteins via NDPK B/Gß{gamma} complexes requires the intermediate phosphorylation of G protein ß subunits at His-266. The mutation of this single amino acid blocks this pathway, followed by reduced myocyte cAMP levels, phospholamban phosphorylation and contraction amplitudes. Our results therefore highlight a novel role of NDPK B as a mammalian G protein-histidine kinase that regulates cAMP synthesis in and contractility of cardiomyocytes by a mechanism bypassing the receptor-induced GDP/GTP exchange at G{alpha}s.


*    Acknowledgments
 
Sources of Funding

This work was supported grants of the Deutsche Forschungsgemeinschaft to T.W. (Wi1373/9-1 and -2) and F.N. (Ni327/5-1); H.J.H. was supported by the Young Medical Investigator Award by the Medical Faculty, University of Heidelberg.

Disclosures

None.


*    Footnotes
 
*F.N. and. T.W. share senior authorship. Back

Original received July 18, 2006; resubmission received December 14, 2006; revised resubmission received March 3, 2007; accepted March 7, 2007.


*    References
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 *References
 

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