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Am J Physiol Heart Circ Physiol 294: H1335-H1347, 2008. First published January 11, 2008; doi:10.1152/ajpheart.00584.2007
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Enhanced calcium cycling and contractile function in transgenic hearts expressing constitutively active G{alpha}o* protein

Ming Zhu,1,2 Agnieszka A. Gach,2 GongXin Liu,1,2 Xiaomei Xu,2 Chee Chew Lim,3 Julie X. Zhang,2 Lan Mao,4 Kurt Chuprun,5 Walter J. Koch,5 Ronglih Liao,2,3 Gideon Koren,1,2 Burns C. Blaxall,6 and Ulrike Mende1,2

1Division of Cardiology, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, Rhode Island; 2Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; 3Boston University School of Medicine, Boston, Massachusetts; 4Department of Medicine, Duke University Medical Center, Durham, North Carolina; 5Thomas Jefferson University, Philadelphia, Pennsylvania; and 6University of Rochester, Rochester, New York

Submitted 18 May 2007 ; accepted in final form 5 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In contrast to the other heterotrimeric GTP-binding proteins (G proteins) Gs and Gi, the functional role of Go is still poorly defined. To investigate the role of G{alpha}o in the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of G{alpha}o1* (G{alpha}o*), the predominant G{alpha}o isoform in the heart. G{alpha}o expression was increased 3- to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in G{alpha}o* transgenic mice in vivo, along with increased L-type Ca2+ channel current density, calcium transients, and cell shortening in ventricular G{alpha}o*-expressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major Ca2+ handling proteins were largely unchanged, except for a modest reduction in Na+/Ca2+ exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6- and 1.9-fold, respectively, in G{alpha}o* ventricles. Density and affinity of β-adrenoceptors, cAMP levels, and PKA activity were comparable in G{alpha}o* and wild-type myocytes, but protein phosphatase 1 activity was reduced upon G{alpha}o* expression, particularly in the vicinity of the ryanodine receptor. We conclude that G{alpha}o* exerts a positive effect on Ca2+ cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key Ca2+ handling proteins in hearts with constitutive G{alpha}o activation.

G proteins; signal transduction; calcium; contraction; transgenic mice

CARDIAC CONTRACTILE FUNCTION is determined by the intrinsic contractile properties of the heart and is subject to neurohumoral regulation. The main receptors involved in regulating contraction are prototypical G protein-coupled receptors (42). They activate heterotrimeric G proteins that are comprised of {alpha}-, β-, and {gamma}-subunits (40). G proteins are classified according to their {alpha}-subunits, because they primarily determine downstream signaling specificity. Several different G proteins are expressed in the heart (53). Members of the Gs and Gi/o subfamilies play a key role in transmitting extracellular signals that regulate myocyte cell shortening (42): 1) activation of Gs protein-coupled receptors (such as β-adrenergic receptors) leads to increased adenylyate cyclase activity and cAMP and PKA activation. PKA then phosphorylates several Ca2+ handling and contractile proteins, resulting in increased contraction and relaxation. 2) Activation of cardiac receptors that are coupled to members of the pertussis toxin-sensitive Gi/o subfamily (such as A1 adenosine and M2 muscarinic receptors) negatively regulate contractile function in mammalian ventricles in the presence of elevated cAMP ("accentuated antagonism"). While G{alpha}i has the capacity to directly inhibit major cardiac adenylyate cyclase isoforms (47), the functional role of G{alpha}o is not well understood.

Go is an extremely abundant protein in the nervous system (22). Targeted deletion of both isoforms of G{alpha}o in mice lead to altered potassium and calcium channel regulation in neuronal cells and neurological abnormalities (19, 23, 46, 49). In the heart, Go is expressed at a much lower level than in the brain. In fact, its existence in the myocardium was long questioned: it was generally believed that G{alpha}o detected in cardiac tissue originated from Go-rich neuronal cells, until G{alpha}o expression was unequivocally shown in cardiac myocytes (15, 54, 55). Deletion of both G{alpha}o isoforms in the heart was associated with a lack of muscarinic regulation of L-type calcium channels (LCC) in ventricular myocytes (49). Because of a concomitant reduction in Go β{gamma}-subunits upon G{alpha}o deletion (37, 49), relative contributions of {alpha}- and β{gamma}-subunits of Go could not be distinguished in this model.

The functional roles of β{gamma}-subunits that are released from Go proteins cannot easily be tested: selectivity in G{alpha}β{gamma} heterotrimer formation and function appears to exist (20), but the exact isoform composition of Go and other G proteins is still not known. Transgenic mouse models with a global reduction of functionally active Gβ{gamma} (18, 25) have given valuable insights into the overall significance of Gβ{gamma}-mediated signaling in the heart but do not distinguish between different Gβ{gamma} sources or combinations.

The goal of the present study was to gain more insight into the functional role of G{alpha}o for Ca2+ regulation and contractile function in the heart. We generated transgenic mice with cardiac-specific expression of a Q205L point mutant (G{alpha}o*) of the G{alpha}o1 isoform, which is predominant in the heart (34, 44). This mutant lacks functional GTPase activity and is therefore not subject to normal inactivation. It is constitutively active and interacts with downstream effector(s) independent of receptor activation.

We demonstrate enhanced cardiac contractile function in mice expressing G{alpha}o* that was evident under basal conditions and maintained in the presence of isoproterenol. Potential mechanisms were explored by assessing the effect of constitutive G{alpha}o* activation on myocyte shortening, Ca2+ handling, and cell signaling. The results from this study advance our understanding and appreciation of the complexity of Go-mediated signaling and its potential significance for the regulation of cardiac contractile function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of transgenic mice. The 1.1-kb cDNA fragment for constitutively active rat G{alpha}o1 Q205L (G{alpha}o*) was excised with SmaI and HindIII and ligated into the blunted SalI and HindIII sites of a pGEM-9Zf vector containing the murine {alpha}-myosin heavy chain promoter ({alpha}-MHC, 5.5 kb; Ref. 45) and a simian virus 40 intron/polyadenylation signal. The resulting plasmid was confirmed by restriction mapping and nucleotide sequencing. A 7.4-kb linear cDNA fragment containing the {alpha}-MHC promoter, coding sequence for G{alpha}o*, SV40 intron/poly(A) was released with SfiI and NotI digestion. The cDNA fragment was microinjected into pronuclei of fertilized FVB mouse oocytes and implanted into pseudopregnant females. Five independent founders were identified by Southern blot and PCR using probes or primers specific for the G{alpha}o1* cDNA. Transgenic mice and wild-type littermates from lines G{alpha}o*68 and G{alpha}o*69 were used in this study at the age of 6–8 mo. All procedures involving animals were approved by the institutional animal care and use committee of Rhode Island Hospital.

Northern blot analysis. Total RNA was extracted from ventricular and other tissues using RNAzol B (Tel-Test, Friendswood, TX), size-fractionated, transferred to nylon membranes, and hybridized with a 32P-labeled G{alpha}o* cDNA probe that does not cross react with endogenous G{alpha}o mRNA. 32P-labeled GAPDH cDNA and [{gamma}32P]ATP-labeled 18S oligonucleotide probes were used to verify equal loading.

In vivo hemodynamic measurements. Mice were anesthetized with ketamine (100 mg/kg of body wt) and xylazine (2.5 mg/kg of body wt) and were analyzed as described previously (25). Briefly, after endotracheal intubation, mice were connected to a rodent ventilator with supplemental isoflurane anesthesia as necessary. After bilateral vagotomy, a 1.4-Fr high-fidelity micromanometer catheter (Millar Instruments) was inserted into the left carotid artery, which was advanced through the aortic valve to the left ventricle. Hemodynamic measurements were recorded at baseline and 45 s after injection of incremental doses of isoproterenol delivered via the left jugular vein. Isoproterenol doses were specifically chosen to maximize the contractile response but limit the increase in heart rate. Ten sequential beats were averaged for each measurement.

Isolation of ventricular cardiomyocytes. Myocytes from adult mice were obtained by Langendorff-perfusion with Ca2+-free Tyrode's solution containing 126 mM NaCl, 4.4 mM KCl, 1 mM MgCl2, 4 mM Na2HCO3, 10 mM HEPES, and 11 mM glucose (pH 7.3), supplemented with 30 mM 2,3-butanedione monoxime (BDM). After a 3- to 5-min perfusion with noncirculating Tyrode's solution, the heart was perfused for 6 min with recirculating Tyrode's solution containing collagenase I (0.9 mg/ml, Worthington) and 25 µM CaCl2. The flaccid heart was removed from the cannula, the atria was trimmed away, and the ventricular tissue was minced in Tyrode's solution containing 0.1 mM CaCl2 and 2% bovine serum albumine and then incubated for 10 min at 37°C. The majority of the cells was released by titruation and filtered through a 250-µm nylon mesh. The cell suspension was centrifuged at 420 g for 2 min and then gradually subjected to Tyrode's solution with increasing concentrations of calcium and decreasing concentrations of BDM (final: 1 mM CaCl2, no BDM). Typical yields were 1.5–2.5 x 106 cells per heart with 70–80% of the cells retaining their rod-shaped morphology.

L-type Ca2+ channel currents. L-type Ca2+ channel currents (ICa-L) was measured in the absence of Na+ and K+ using whole cell patch clamp recording as previously described (28). ICa-L was activated by a series of 200-ms depolarization pulses from –50-mV holding potential to test potentials ranging from –40 to +60 mV in 2-s intervals. The bath solution contained (mM): 135 tetraethylammonium-Cl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH adjusted to 7.4 with tetraethylammonium-OH. The pipette solution contained (mM): 120 CsCl, 5 Mg-ATP, 10 EGTA, 1 MgCl2, and 10 HEPES, with pH 7.2 adjusted with CsOH. The current at each potential was normalized to cell capacitance, and the peak current-voltage relationship was used for data evaluation. Voltage dependence of ICa-L activation was fit with Boltzman equation G/Gmax = 1/[1 + exp(V0.5-V)/k] with V0.5 as half-maximal GCa and k as slope factor.

Ca2+ transients and mechanical properties. Ca2+ transients and mechanical properties were assessed in freshly isolated ventricular myocytes, which were continuously perfused in a heated chamber (36 ± 1°C) with perfusion buffer containing (in mM): 137 NaCl, 5.4 KCl, 0.5 MgCl2, 10 HEPES, 5.5 glucose, 1.2 CaCl2, and 0.5 probenecid and electrically stimulated at 0.5 Hz. Single myocytes included in the study were selected according to the following criteria: 1) rod shaped with a clear striation pattern, 2) quiescent when unstimulated, and 3) stable mechanical behavior at 0.5 Hz and 36°C for at least 3 min. No cells were used more than 6 h after isolation. The cells were allowed to equilibrate for 3 min before the perfusion buffer was switched to include isoproterenol (0.1 µM). The response to isoproterenol reached a steady state within 5 min after switching the perfusate. Only data from matched pairs were included in the analysis. Cell shorting was monitored by a digital edge detection system (IonOptix) with a sampling rate of 240 Hz. Ca2+ transients were measured in fura-2/AM loaded cardiomyocytes (1 µM for 15 min at room temperature) using a dual excitation Hyperswitch (IonOptix). For each cell, background fluorescence was recorded at four points adjacent to the myocyte. For both steady-state twitches and intracellular transients, 10 traces were averaged and the common kinetic and amplitude parameters were extracted by software (Ionwizard, IonOptix). Sarcoplasmic reticulum (SR) Ca2+ content was assessed by acquiring intracellular calcium concentration ([Ca2+]i) in myocytes in which prior electrical stimulation for 30 s was ceased 5 s before application of 15 mM of caffeine.

Myofilament calcium sensitivity. Myofilament calcium sensitivity was assessed in Triton-permeabilized ventricular myocytes according to a previously described protocol with minor modifications (27). Briefly, freshly isolated myocytes were perfused in the calcium-containing perfusion buffer described in Ca2+ transients and mechanical properties with or without 0.1 µM isoproterenol for 5 min. Myocytes were subsequently permeabilized or skinned in calcium-free relaxing solution (in mM): 10 EGTA, 5.9 MgAc, 5.9 Na2ATP, 10 creatine phosphate, 40 imidazole, 70 potassium proprionate, 5 NaN3, 1 DTT, 0.5 PMSF, 0.04 leupeptin, and 50 U/ml creatine phosphokinase, pH 7.0 containing 1% Triton X-100. To assess myofilament calcium sensitivity, skinned myocytes were exposed to increasing calcium-containing activating solutions (from pCa 7 to pCa 5.3), returning to calcium-free relaxing solution (pCa 9) between each activating solution. The pCa solutions were made by appropriately adding calcium proportionate to the relaxing solution according to the calculations of Fabiato (16). Rapid solution switching was achieved using a Fast-Step perfusion system (Warner Instruments), and sarcomere length was continuously measured using the IonOptix system as described previously (27).

Western blot analysis. Ventricular tissue pieces were homogenized and, where indicated, ultracentrifuged (48,500 g) to obtain membrane and cytosolic fractions as described previously (35). Protein was measured using the Bradford microassay (Bio-Rad) with BSA as standard. Equal amounts of membrane and cytosolic protein were separated on 10% (G protein subunits and PKC{alpha}) or crude extracts on continuous 4–12% and 4–20% gradient gels (Ca2+ handling proteins) and transferred to nitrocellulose membranes. After transfer, each gel was stained with Coomassie blue R-250 to verify complete protein transfer. The nitrocellulose membranes were stained with Ponceau S to confirm equal loading and even transfer efficiency. Immunoblots were performed as described previously (36) using a rabbit polyclonal antibody against affinity-purified bovine brain G{alpha}o (1:250; Ref. 57); rabbit polyclonal antibodies against G{alpha}s (sc-383, 1:1,000, Santa Cruz Biotechnology), G{alpha}i (AS/7, 1:1,000, NEN Life Science Products), G{alpha}q/11 (C-19, 1:1,000, Santa Cruz Biotechnology), and Gβ (βcommon, 1:1,000, Upstate Biotechnology); a goat polyclonal antibody against SR Ca2+-ATPase (SERCA2a, sc-8094, 1:100, Santa Cruz Biotechnology); mouse monoclonal antibodies against phospholamban (PLB; MA3-922, 1 µg/ml) and Na+/Ca2+ exchanger (NCX1; MA3-926, 1:200, all from Affinity BioReagents); and rabbit polyclonal antibodies against the LCC {alpha}1c subunit (ACC-013, 1:200, Alomone labs), calsequestrin (PA1-913, 1:2,500), and phosphorylated phospholamban (p-PLB, at Ser16, 1:1,000, Research Diagnostics). The specificity of p-PLB antibody was confirmed by in vitro phosphorylation of cardiac crude homogenates with PKA (data not shown). Proteins of interest were visualized by chemiluminescence (SuperSignal West Pico Substrate, Pierce) using horseradish-conjugated secondary antibodies (ImmunoPure, Pierce). Film exposure times varied depending on the abundance of the protein and were adjusted so that the signal stayed within the linear range. Quantitation was done by computerized densitometry on scanned blots using the National Institutes of Health Image 1.61/ppc. The average of arbitrary units obtained in wild-type samples was set at 100% to which both individual transgenic and wild-type samples were then normalized.

Assessment of ryanodine receptor. Expression and phosphorylation were performed as described previously (33). Ryanodine receptor (RyR2) was immunoprecipitated from ventricular homogenates by incubating 500 µg of homogenate with an anti-RyR antibody in 0.5 ml of a modified RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 0.9% NaCl, 1.0 mM NaF, 1.0 mM Na3VO4, 0.25% Triton X-100, and protease inhibitors overnight at 4°C. Protein A-Sepharose beads were added and incubated at 4°C for 1 h. The immunoprecipitated proteins were then separated by SDS-PAGE and analyzed for RyR2 expression and phosphorylation by immunoblotting using antibodies against RyR2 and phosphorylated RyR2 (at Ser2809). Western blots of immunoprecipitated RyR2 were also probed with monoclonal mouse antibodies against PKA, protein phosphatase 2A (PP2A), and protein phosphatase 1 (PP1; all used at 1:2,000, Transduction Laboratories).

Binding assays for β-adrenoceptors. Adult mouse ventricular myocyte membranes were prepared as described previously (1) with minor modifications. Briefly, cells were homogenized by hand using a glass-on-glass dounce in buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and protease inhibitors. The homogenate was centrifuged at 800 g for 10 min, and the supernatant was transferred to a new tube. The low speed pellet was resuspended and homogenized a second time and spun as above, and like supernatants were centrifuged at 100,000 g for 1 h. The pellet, taken as the crude plasma membrane fraction, was resuspended in binding buffer (75 mM Tris-HCl, pH 7.4 at 37 °C), 12.5 mM MgCl2, and 2 mM EDTA and stored at –80 °C until use in radioligand binding experiments. Protein concentration was measured using the Bradford assay (Bio-Rad) with BSA as standard.

The β-adrenergic receptors were quantified using [125I]iodocyanopindolol (Amersham) in saturation binding experiments as described previously (1) with minor modifications. Briefly, in duplicate, 20 µg of membrane protein per reaction were incubated with increasing final concentrations of 125I-labeled iodocyanopindolol ranging from 1 to 300 pM in binding buffer. Nonspecific binding was defined as binding in the presence of 100 µM alprenolol (Sigma). After 1 h at 37 °C, reactions were terminated by rapid filtration using a Brandel Model M48 cell harvester through GF/B filters and washed thoroughly using 10 mM Tris-HCl and 10 mM EDTA buffer. The filters were transferred to tubes and counted in a gamma counter for 1 min. Maximal number of binding sites (Bmax) and radioligand binding affinity (Kd) were determined using GraphPad Prism.

cAMP determinations. Freshly isolated ventricular mouse myocytes were adjusted to 1 x 105 in 500 µl buffer containing 126 mM NaCl, 4.4 mM KCl, 1 mM MgCl2, 4 mM NaHCO3, 10 mM HEPES, and 11 mM glucose and incubated in 1 mM IBMX at 37°C, followed by isoproterenol stimulation for 30 min at submaximal concentration (previously determined with a dose-response curve; data not shown). The reaction was stopped by addition of 50 µl ice-cold 20% perchloric acid. After vortexing and centrifugation at 12,000 g for 10 min, the pellets were stored at –80°C for subsequent protein determination using the DC assay system (Bio-Rad). The supernatants were adjusted to pH 4–5 with 20% KHCO3 (~50 µl), and the resulting precipitate was centrifuged for 10 min at 12,000 g. cAMP levels were measured in the supernatants using an enzyme immunoassay (Cayman Chemicals) that is based on competition between free cAMP (in the sample) and a cAMP tracer (linked to an acetylcholinesterase molecule) for a limited number of cAMP-specific rabbit antiserum binding sites. It was conducted in 96-well coated with a mouse monoclonal anti-rabbit antibody. After overnight incubation at 4°C, followed by washing of all unbound reagents, Ellman's reagent containing a substrate for acetylcholinesterase was added to each well and incubated at room temperature for ~40 min. The color intensity of the reaction product was measured spectrophotometrically at 420 nm. Each sample was assayed in duplicate, calibrated against a standard curve, and expressed in picomoles of cAMP per milligrams of protein.

PKA activity assay. Freshly isolated ventricular mouse myocytes were divided into aliquots containing 2 x 105 myocytes, briefly washed with PBS and extraction buffer (see below), flash-frozen, and stored at –80°C. On the day of the assay, myocyte pellets were homogenized in ice-cold extraction buffer containing 25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 10 mM β-mercaptoethanol, and proteinase inhibitors. After a 5-min centrifugation at 14,000 rpm at 4°C, supernatants were collected and assayed using the SignaTECT cAMP-dependent PKA Assay System (Promega) according to the manufacturer's instructions. Each sample was assayed in duplicate at three different dilutions to stay within the linear range that was previously determined using different cell numbers and dilutions. The final reaction mixture contained 40 mM Tris-HCl (pH 7.4), 20 mM MgCl2, 0.125 mg/ml BSA, 5 µM cAMP, 100 µM ATP (containing 0.5 µCi [{gamma}-32P]ATP, 3,000 Ci/mmol), and 100 µM PKA biotinylated peptide substrate. After incubation for 5 min at 30°C, the reaction was terminated by addition of guanidine hydrochloride (2.5 M final), and the reaction products were spotted onto a streptavidin matrix (SAM2 Biotin Capture Membrane). After being washed with 2 M NaCl in the absence and then presence of 1% H3PO4, followed by a wash in deionized water, [{gamma}-32P]ATP incorporated into the biotinylated peptide substrate was determined by scintillation counting. PKA enzyme activity was normalized to protein (assessed by the Bradford protein assay, Bio-Rad) and expressed in picomoles of ATP per minute per micrograms of protein.

Protein phosphatase activity. Protein phosphatase activity was determined by measuring the release of inorganic phosphate from a 32P-labeled substrate using a protein serine/threonine phosphatase activity assay system (Biolabs). First, myelin basic protein (MyBP, 0.2 mM) was radioactively labeled overnight in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Brij 35, 1,250 U/ml PKA, 1 mM ATP, and 0.25 µCi/µl [{gamma}-32P]ATP (3,000 Ci/mmol). TCA (1/9 vol of 100%) was added to precipitate the phosphoprotein, inactivate PKA, and remove excess ATP. 32P-labeled MyBP was purified from residual ATP by washing with 20% TCA and dialysis for 16 h at 4°C with buffer containing 25 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 2 mM DTT, and 0.01% Brij 35. Scintillation counting was used to determine the radioactivity and calculate the incorporated phosphate concentration (µM).

On the day of the assay, flash-frozen ventricular myocytes were homogenized in lysis buffer containing 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 0.01% Brij 35, and proteinase inhibitors and centrifuged at 2,800 rpm for 5 min at 4°C to collect crude extract (supernatant). Protein concentrations were determined (Bradford protein assay, Bio-Rad). Equal amounts of protein (400 ng protein/reaction) were mixed with 32P-labeled MyBP (30 µM) in buffer containing 50 mM Tris-HCl (pH 7), 0.1 mM EDTA, 5 mM DTT, and 0.01% Brij 35 and then were incubated at 30°C for 10 min. The reaction was terminated with TCA (final 16%) and centrifuged at 12,000 g for 5 min, and the supernatants were collected for scintillation counting. Phosphatase activity was expressed in nanomoles of [32P]Pi released per minute per milligrams of protein. Each sample was assayed at three different dilutions in duplicates within the linear range of the assay. A low concentration of ocadaic acid (OA; 1 nM) was used to distinguish PP2A from total phosphatase activity. OA at 0.5 µM (and inhibitor 2 at 0.1 µM) was used to distinguish PP1{alpha} activity.

Phosphatase activity associated with RyR2 complex was measured as follows: protein A beads containing the immunoprecipitated RyR complex were washed three times with ice-cold Ser/Thr assay buffer (50 mM Tris-HCl, pH 7.0, and 0.1 mM CaCl2). PP1 or PP2A activity was assayed using a Ser/Thr phosphatase assay kit (Upstate Biotechnology). RyR2 complex beads in 50 µl of Ser/Thr assay buffer were incubated with the phosphopeptide KRpTIRR at 30°C for 30 min. The beads were pelleted, and 25 µl of supernatant were analyzed for free phosphate by dilution with 100 µl of developing solution (malachite green). After incubation for 15 min, the release of phosphate was quantified by measuring the absorbance at 620 nm in a microtiter plate reader. PP1 activity in the complex was determined from the phosphatase activity inhibited by 100 nM protein phosphatase inhibitor 2, and PP2A activity was determined by the phosphatase inhibited by 1 nM OA.

Adenoviral infection of cardiomyocytes. The cDNA encoding G{alpha}o1 Q205L (see above) was amplified using PCR forward primers suitable for directional TOPO cloning (CACC overhang). They contained either GGA or GCA (encoding glycine or alanine, respectively) after the start codon. PCR products were introduced into the Gateway pENTR vector (Invitrogen) using topoisomerase I. Sequences and proper integration were confirmed, and the constructs were transferred in a lambda phage site-specific recombination reaction with >90% efficiency (data not shown) into an adenoviral destination vector. It contained two attR DNA segments downstream of a bicistronic cytomegalovirus promoter for green fluorescent protein coexpression (provided by the Harvard Gene Therapy Initiative). Adenoviral DNA was amplified, linearized, and infected into HEK293 cells. Adenoviruses were amplified, harvested, purified, and titered using standard techniques.

After isolation, ventricular myocytes were plated on laminin-coated (10 µg/ml) culture dishes and maintained in "defined medium" (14). After 24 h, myocytes were incubated in 0.5 ml serum-free DMEM containing sufficient adenovirus to achieve a multiplicity of infection of 50, which was previously determined to lead to >90% infection efficiency (data not shown). After 2 h, the total volume was brought up to 2 ml per well and the myocytes were then incubated for 48 h at 37 °C. Myocytes were then either lysed directly or collected for subsequent membrane and/or cytosol fractionation at 100,000 g.

Statistical analysis. Data are reported as mean (SD) for number (n) of determinations. Statistical analyses between wild-type and G{alpha}o* mice were performed using paired or unpaired, two-tailed Student's t-tests. Two-way ANOVA (with repeated measures where appropriate) was used when two different variables were assessed. Microsoft Excel and GraphPadPrism software (Version 4) was used for statistical analysis. A P value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic mouse model. After verifying by RT-PCR that G{alpha}o1 was the predominant G{alpha}o isoform in adult mouse ventricular myocytes (data not shown), we generated transgenic mouse lines using the {alpha}-MHC promoter to achieve cardiac-specific expression of constitutively active G{alpha}o1 Q205L (G{alpha}o*; Fig. 1A, left). Transgenic G{alpha}o* mRNA (Fig. 1A, right) and protein levels (Fig. 1B) varied among five independent transgenic lines. The corresponding increase in the total amount of ventricular G{alpha}o protein compared with wild-type controls ranged from 3- to 15-fold (Fig. 1C). In contrast to wild types, G{alpha}o was found in membrane and cytosolic fractions from transgenic ventricles (Fig. 1B). The relative distribution between membrane-bound and cytosolic G{alpha}o (25–30 vs. 70–75%) was comparable in all lines despite marked differences in the overall expression level of G{alpha}o (Fig. 1C). G{alpha}o expression and distribution were stable over time, as shown for line 68 in Fig. 1B, bottom. Importantly, the expression of {alpha}-subunits from the other major cardiac G protein subfamilies (G{alpha}i, G{alpha}s, and G{alpha}q) and β-subunits (tested with a βcom antibody that recognizes all β-subunit expressed in the heart) was comparable in membrane fractions from G{alpha}o*68 and wild-type ventricles (Fig. 1D). These G protein subunits were not detectable in the cytosol (data not shown).


Figure 1
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  		Fig. 1. G{alpha}o mRNA and protein and other G protein expression. A: Northern blots of 10 µg total RNA from different G{alpha}o*68 tissue (left; H, heart; Br, brain; Li, liver; Sp, spleen; Lu, lung; Sm, skeletal muscle) or ventricular RNA from 5 independent transgenic lines (right). Both blots were probed with a G{alpha}o* cDNA probe, which does not cross react with endogenous G{alpha}o mRNA. B: Western blots of ventricular membrane (Mem; M) and cytosolic (Cyt; C) protein (120 µg/lane). G{alpha}o expression at 2 mo in 4 transgenic lines (L69, L72, L67, and L70) and wild types (wt; top; n = 2 each; Ctr, rat brain crude homogenate). Time course of ventricular G{alpha}o expression in line 68 (bottom). Both blots were probed with an affinity-purified G{alpha}o antibody that recognizes endogenous and transgenic G{alpha}o protein. C: G{alpha}o expression and membrane-cytosol distribution in transgenic compared with wild-type ventricles (n = 3 each). Arbitrary units were obtained by densitometry of Western blots with equal amounts of membrane and cytosolic proteins and then multiplied with the amount of total protein in each fraction. These values were normalized to wild-type controls (set at 1). D: Western blots of ventricular membranes from 2-wk-old and 2- and 6-mo-old G{alpha}o*68 mice (L68) and age-matched wild types probed with indicated antibodies. G{alpha}s-l and G{alpha}s-sh denote the long and short isoform of G{alpha}s, respectively.

 
Mice from line 68 (G{alpha}o*68 or L68) were used for detailed phenotypic analysis in this study; key results were confirmed in mice from line 69 (G{alpha}o*69 or L69). Transgenic mice had a normal life span with no difference in heart and/or body weight or apparent changes in gross or histological morphology (data not shown). No significant gender differences were observed (data not shown).

Contractile function. In vivo hemodynamic measurements revealed that left ventricular systolic pressure and maximal rate of contraction and relaxation were significantly increased under basal conditions in G{alpha}o*68 hearts compared with controls (Fig. 2, A and B). These differences were maintained in the presence of increasing doses of the β-adrenergic agonist isoproterenol, suggesting that the actual sensitivity to β-adrenergic stimulation was unchanged (see below). Both basal heart rates and the chronotropic response to isoproterenol were comparable in anesthetized G{alpha}o* and wild-type mice (Fig. 2C). Heart rates in conscious mice were in the normal range (675–735 bpm) and also comparable between transgenic and wild-type mice (data not shown).


Figure 2
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  		Fig. 2. In vivo assessment of left ventricular (LV) function. Parameters of left ventricular systolic function [A; LV systolic pressure (LVSP) and maximal rate of contraction (+dP/dt)] and diastolic function [B; LV end-diastolic pressure (LVEDP) and maximal rate of relaxation (–dP/dt)] and heart rate (C) in male G{alpha}o*68 (bullet, n = 7) and wild-type ({circ}, n = 8) mice at baseline and in response to increasing doses of isoproterenol (50, 500, and 1,000 pg). LVSP, +dP/dt, and –dP/dt were significantly increased under basal conditions in G{alpha}o*68 hearts compared with controls (P < 0.005, P < 0.02, and P < 0.05, respectively). P values (2-way ANOVA for repeated measures) for G{alpha}o*68 and wild-type isoproterenol dose- response curves were P < 0.001 (LVSP), P < 0.001 (+dP/dt), P < 0.01 (–dP/dt), and P = 0.06 (LVEDP).

 
We next determined whether increased cardiac contraction was due to changes in the myocyte cell shortening and/or increased sympathetic regulation. Percentages of cell shortening as well as maximal rates of cell shortening and relengthening were significantly enhanced in transgenic myocytes under basal conditions (Table 1). Each of these parameters was also higher in G{alpha}o*68 myocytes after isoproterenol stimulation: the differences compared with wild-type myocytes were proportional to the relative differences observed at baseline but were not statistically significant. Bmax and Kd of β-adrenoceptors were both comparable in G{alpha}o*-expressing and wild-type myocytes [Bmax in fmol/mg protein: 26.9 (SD 5.2) vs. 24.0 (SD 7.5), P = 0.595; Kd in pM: 44.9 (SD 29.6) and 43.6 (42.1), P = 0.967; n = 3–4 each]. Together, these results suggest that enhanced cardiac contractile function in the transgenic mice was mainly due to enhanced myocyte shortening. Similar changes were obtained in myocytes from G{alpha}o*69 mice (data not shown).


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  		Table 1. Twitch and Ca2+ transient parameters

 
Intracellular Ca2+ transients and myofibrillar sensitivity. We next addressed whether the increase in myocyte shortening was due to corresponding changes in calcium transients and/ or altered myofilament Ca2+ sensitivity. The same myocytes used to assess cell shortening were labeled with fura-2/AM (Table 1): diastolic fura-2 ratios were comparable but transgenic myocytes showed a 40% increase in peak [Ca2+]i amplitude under basal conditions. The relative difference at baseline was again maintained in the presence of isoproterenol. In contrast, myofilament Ca2+ sensitivity was comparable in transgenic and wild-type myocytes both at basal and after β-adrenergic stimulation (Fig. 3A). This was shown with an assay system that is capable of detecting rightward shifts in sarcomere-pCA relationships in PKA- or isoproterenol-treated myocytes (Fig. 3B). Thus, a rise in [Ca2+]i was likely responsible for increased cell shortening in G{alpha}o*68-expressing myocytes. To better understand the underlying mechanism(s), we examined three key determinants of Ca2+ handling in cardiac myocytes.


Figure 3
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  		Fig. 3. Myofilament Ca2+ sensitivity. A: plot of sarcomere length vs. pCa under basal conditions (left) and after stimulation with isoproterenol (0.1 µM for 5 min; right) in skinned myocytes from G{alpha}o*68 (bullet) and wild-type ({circ}) mice; n = 14–17 cells from 2 different animals in each group. B: effect of PKA (2 U/µl for 30 min at room temperature after skinning; left; bullet) and isoproterenol (Iso; 0.1 µM for 5 min at room temperature before skinning; right; bullet) on the sarcomere length/pCa relationship compared with respective vehicle controls ({circ}). Compared with vehicle, P values (2-way ANOVA for repeated measures) for pCA dose-response curves in PKA- or Iso-treated wild-type myocytes were P < 0.0005 (n = 6–8) and P < 0.05 (n = 3), respectively.

 
Calcium handling. One important determinant of [Ca2+]i amplitude is the amount of Ca2+ entering the myocyte via LCC, which in turn triggers Ca2+ release from the SR. We therefore assessed LCC currents by measuring peak inward ICa-L amplitudes and found that peak ICa-L current density was significantly increased in G{alpha}o*-expressing myocytes under basal conditions (Fig. 4, A and B, left); this relative increase was maintained in the presence of isoproterenol (right). Voltage-dependent activation curves were shifted to a more negative potential under both conditions (Fig. 4C, left and right). The voltage at which GCa was half of its maximally activated value (V0.5) was –9.9 (SD 3.3) mV in G{alpha}o*68 vs. –3.1 (SD 7.5) mV in wild type at basal (P = 0.007; n = 9–11 each) and –12.2 (SD 2.3) mV in G{alpha}o*68 vs. –7.0 (SD 6.9) mV in wild types in the presence of isoproterenol (P = 0.015; n = 9–11).


Figure 4
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  		Fig. 4. L-type Ca2+ channel currents (ICa-L). A: representative recordings of whole cell ICa-L in wild-type (top) and G{alpha}o*68 (bottom) cardiomyocytes under basal conditions (left) and in the presence of isoproterenol (0.1 µM; right). The current at each potential was normalized to cell capacitance. Insets: calibration and voltage protocol. B: averaged ICa-L peak current-voltage relationship from 9 wild-type cells (from 2 hearts, {circ}; {square}) and 11 G{alpha}o*68 cells (from 2 hearts, bullet; {blacksquare}) under basal conditions (left) and in the presence of isoproterenol (0.1 µM, right). P values (2-way ANOVA) for G{alpha}o*68 vs. wild-type were P < 0.0004 (basal) and P < 0.005 (Iso). C: voltage-dependence of ICa-L activation. See B for details. P values (2-way ANOVA) for G{alpha}o*68 vs. wild-type were P < 0.004 (basal) and P < 0.003 (Iso). G/Gmax= 1/[1+exp(V0.5-V)/k] with V0.5 as half-maximal GCa and k as slope factor.

 
Other important determinants of [Ca2+]i amplitude and kinetics are the expression and activity of proteins that regulate SR Ca2+ cycling. Expression levels of the SR Ca2+ binding protein calsequestrin and SERCA2a, which pumps Ca2+ back into the SR during diastole, were comparable in ventricular homogenates from transgenic and wild-type mice (Fig. 5A). Only the NCX1 was slightly reduced (Fig. 5A). Although neither RyR2 (the major SR Ca2+ release channel) nor PLB (a negative regulator of SERCA2) were changed in their expression (Fig. 5, B and C), phosphorylation at their respective PKA sites [i.e., Ser2809 for RyR2 (52) and Ser16 for PLB (21)] was increased 1.6- and 1.9-fold in transgenic ventricles under basal conditions. To test whether this effect was due to membrane-bound or cytosolic G{alpha}o*, we expressed G{alpha}o* and its myristoylation-deficient mutant (G{alpha}o*G2A; Ref. 38) in adult rat ventricular myocytes via adenoviral gene transfer. G{alpha}o* was primarily detected in the membrane fraction, whereas as expected G{alpha}o*G2A was targeted to the cytosol (Fig. 6A). Only myocytes expressing membrane-bound G{alpha}o* showed increased PLB Ser16 phosphorylation compared with empty adenovirus control (Fig. 6B). Due to limited starting material, RyR2 phosphorylation could not be assessed.


Figure 5
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  		Fig. 5. Ca2+ handling protein expression and phosphorylation. A, left: Western blots with increasing amounts (2.5–10 µg/lane) of G{alpha}o*68 and wild-type ventricular homogenates were probed with antibodies recognizing the indicated Ca2+ handling proteins. Right: group data from G{alpha}o*68 and wild-type ventricles (n = 3–5). #P < 0.01 vs. wild type. SERCA2a, sacroplasmic reticulum Ca2+-ATPase; NCX1, Na+/Ca2+ exchanger; CSQ, calsequestrin; LCC, L-type calcium channels. B: Western blots (top) and group data (bottom; n = 7 each) for ventricular homogenates from wild-type and G{alpha}o*68 mice that were immunoprecipitated with an antibody recognizing the ryanodine receptor (RyR2). Western blots of the resulting pellets were probed with the indicated antibodies. #P < 0.01 vs. wild type. C: Western blot (left) and group data (right; n = 4 each) for G{alpha}o*68 and wild-type ventricular homogenates (10 µg) that were probed with antibodies recognizing total and phosphorylated (p) phospholamban (PLB; at Ser16). #P < 0.001 vs. wild type.

 

Figure 6
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  		Fig. 6. In vitro expression of differentially targeted G{alpha}o*. Comparison of PLB Ser16 phosphorylation upon expression of differentially targeted G{alpha}o* in adult rat ventricular myocytes that had been infected in duplicates for 48 h with adenoviruses [50 multiplicity of infection (MOI)] encoding G{alpha}o* or G{alpha}o*G2A lacking the myristoylation site or a control virus. A: Western blot with equal amounts of crude homogenate (CH; 10 µg/lane), cytosolic (5 µg/lane), or membrane (7.5 µg/lane) protein that was probed for G{alpha}o expression. B: Western blot with equal amounts of myocyte cell lysates (20 µg/lane) that were probed for total and Ser16-phosphorylated PLB.

 
Another factor influencing Ca2+ transients is the amount of Ca2+ stored in the SR, which is dependent on both RyR2 and PLB. We indirectly assessed SR Ca2+ load by acquiring caffeine-induced [Ca2+]i and did not detect any difference in caffeine-induced peak [Ca2+]i amplitudes at basal [0.17 (SD 0.07) in wt vs. 0.18 (SD 0.06) in G{alpha}o*; n = 16–18] or in the presence of isoproterenol [0.24 (SD 0.06) in wt vs. 0.25 (SD 0.07) in G{alpha}o*; n = 18–26], suggesting normal SR Ca2+ load.

Taken together, the two major changes in calcium handling in G{alpha}o* transgenic mice were increased Ca2+ influx via the LCC and enhanced RyR2 and PLB phosphorylation.

cAMP-mediated signal transduction. To investigate potential signaling mechanisms that might be involved in RyR2 and PLB hyperphosphorylation, we tested whether PKA-mediated phosphorylation might be enhanced. Neither cAMP accumulation (basal or in response to β-adrenergic stimulation) nor PKA activity were altered in G{alpha}o*-expressing ventricular myocytes (Fig. 7, A and B). To determine if altered dephosphorylation might be involved, we measured total phosphatase activity in myocyte lysates and determined PP1 and PP2A activity using specific inhibitors. They made up 30 (SD 1) and 66 (SD 1) % of total phosphatase activity (n = 3 each), respectively. In G{alpha}o* myocytes, PP2A and PP1 activity was slightly albeit not significantly reduced (Fig. 7C). Taking advantage of the fact that RyR2 can be immunopreciptated together with many of its associated proteins (33), we also examined PP1 and PP2A activity in the direct vicinity of one of their targets. Within the RyR2 complex, PP1 and PP2A phosphatase activity made up 47 (SD 1) and 48 (SD1) % of total phosphatase activity, respectively (n = 6 each). Importantly, RyR2-associated PP1 activity was significantly decreased by 22 (SD 7) % in transgenes (Fig. 7D). This was associated with a 45 (SD 8) % decrease in co-immunoprecipitated PP1 (Fig. 7E). In contrast, the amounts of PP2A and PKA in the RyR2 complex were comparable in transgenes and wild-types (Fig. 7E). We next asked whether a decrease in PKC{alpha} expression or activation might contribute to the observed decrease in PP1 in G{alpha}o* mice, since PKC{alpha} can activate PP1 (6). This does not appear to be the case: total PKC{alpha} expression and its distribution between soluble and particulate fraction were comparable with wild types (data not shown). Together, these findings point towards PP1 as a possible downstream mediator of G{alpha}o*.


Figure 7
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  		Fig. 7. Signal transduction assays. A: cAMP accumulation at baseline and in response to stimulation with isoproterenol (1 µM) in ventricular myocytes from G{alpha}o*68 and wild-type mice (n = 3 each). *P < 0.01 and P < 0.04 for Iso vs. basal in wild type and G{alpha}o*68, respectively. B: PKA activity in ventricular myocytes from G{alpha}o*68 and wild-type mice (n = 3 each). C: global protein phosphatase (PP) 2A and PP1 activity (in nmol [32P]Pi released per min per mg protein) in ventricular myocytes from G{alpha}o*68 and wild-type mice (n = 3 each). D: activity of PP2A and PP1 (pmol per min per mg protein) associated with the RyR2 in G{alpha}o*68 and wild types (n = 6 each). #P < 0.01 vs. wild type. E: Western blots (left) and group data in G{alpha}o*68 and wild-type ventricles (right, n = 4 each) illustrating the relative amount of RyR2-associated PKA, PP2A, and PP1 (see Fig. 5B for corresponding Western blot of immunoprecipitated RyR2). #P < 0.01 vs. wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we developed a new mouse model with cardiac-specific expression of constitutively active G{alpha}o* that allowed us to assess downstream effects of G{alpha}o in the heart without confounding effects from Go β{gamma}-subunits and other G proteins. We demonstrate enhanced contractile function in the transgenic mice in vivo that is likely due to increases in LCC density, calcium transients, and cell shortening that were observed in ventricular G{alpha}o* myocytes. These changes were already evident under baseline conditions and simply maintained under β-adrenergic stimulation. Key biochemical findings were 1) enhanced ventricular phosphorylation of RyR2 and PLB at their respective PKA sites in G{alpha}o* mice that likely contribute to the observed functional phenotype, and 2) reduced PP1 activity (with no change PKA activity) that suggests altered dephosphorylation (rather than phosphorylation) as a potential cause for RyR2 and PLB hyperphosphorylation in this model. Together, these changes associated with cardiac-specific constitutive G{alpha}o activation in vivo advance our understanding of the functional role of Go in the heart.

Experimental model. We chose constitutively active G{alpha}o* for this study, because it has a low affinity for Gβ{gamma} (11) and does not require endogenous Gβ{gamma} or receptors to become activated. The specific advantages were as follows: 1) G{alpha}o* could directly activate downstream effector(s). 2) Signaling effects mediated by Gβ{gamma} subunits upon their release from endogenous G proteins were not hampered. 3) The equilibrium of endogenous G{alpha} subunits would not be shifted to their dissociated, active state. 4) Since Gβ{gamma} subunits appear to be protected from degradation when associated with G{alpha}o (37), G{alpha}o* was unlikely to increase Gβ{gamma} expression as confirmed in Fig. 1D).

Membrane attachment of G{alpha}o is facilitated by N-terminal palmitoylation and myristoylation and its interaction with Gβ{gamma} subunits (9). Activated G{alpha} subunits can have increased palmitoylation turnover (51). In COS-7 overexpression studies, activated and unactivated G{alpha}o had similar subcellular distribution despite a considerable reduction in G{alpha}o* palmitoylation (50). The reason for the cytosolic presence of G{alpha}o* in transgenic hearts is still unclear at present. It was observed in different transgenic lines independent of G{alpha}o* expression levels (Fig. 1C), which argues against a mere artifact of overexpression (e.g., by overwhelming the posttranslational machinery required for proper targeting). Further work is required to define the determinants for subcellular targeting of G{alpha}o* in myocytes and to dissect the functional role of membrane-associated vs. cytosolic G{alpha}o*. Short-term overexpression experiments in isolated myocytes (Fig. 6) showed that only G{alpha}o* with predominant membrane targeting but not G{alpha}o* with predominant cytosol targeting (myristoylation-deficient G2A mutant) leads to PLB hyperphosphorylation, indicating that membrane-bound G{alpha}o* might be the primary mediator. It also suggests that short-term G{alpha}o* expression is sufficient to induce PLB hyperphosphorylation in vitro. Additional transgenic models are needed to test both hypotheses in vivo. First, transgenic mice with cardiac-specific expression of strictly membrane- vs. cytosol-targeted G{alpha}o* will be required to determine the effects of differentially localized G{alpha}o in vivo. Second, an inducible model will be required to determine how the timing and duration of transgenic G{alpha}o expression might affect the phenotype, thereby addressing if and how adaptative processes might be involved.

Phenotypic characterization. One of the major findings in G{alpha}o* transgenic mice was the increase in contractile function in isolated myocytes and in vivo (Table 1 and Fig. 2, respectively). Examination of two independent transgenic lines with different G{alpha}o* expression levels indicated that the G{alpha}o* amount expressed in line 69 was sufficient for phenotype induction and that additional G{alpha}o* did not lead to further enhancement.

Mechanistically, we observed a significant increase in Ca2+ influx through the LCC (Fig. 4). Comparable Western blot signals (see Fig. 5A) argue against changes in {alpha}1c expression as a cause for the increase in ICa-L density. The small negative shift of the voltage dependence of ICa-L activation in G{alpha}o* myocytes compared with wild types (Fig. 4C) would be consistent with increased LCC phosphorylation, but additional studies are needed to substantiate this notion and to determine whether or not changes in the local phosphatase/kinase balance might potentially contribute. Auxiliary subunits could be involved as well. The LCC is subject to complex regulation (24); delineating the mechanism of G{alpha}o*-mediated LCC regulation is beyond the scope of the present study.

The third important finding is increased phosphorylation of two other important calcium handling proteins (RyR2 and PLB) at their respective PKA sites upon G{alpha}o* expression (Fig. 5, B and C). PKA phosphorylation of RyR2 is part of an integrated physiological response that leads to increased excitation-contraction coupling gain and increased cardiac output (52). The target sites and functional consequences of PKA-mediated RyR2 hyperphosphorylation have been the subject of intense debate (2, 4, 26, 31) and remain to be determined in G{alpha}o* mice. PLB phosphorylation at Ser 16 generally does not affect SERCA2a expression (see also Fig. 5A) but relieves the inhibition that unphosphorylated PLB normally exerts on SERCA2a (21). Consistent with this notion, the time constant for the decay of basal Ca2+ transients was significantly decreased in G{alpha}o* myocytes with enhanced PLB Ser16 phosphorylation compared with wild type (Table 1).

All functional effects described above were already evident under basal conditions. Isoproterenol caused only a further proportional increase, indicating that β-adrenergic responsiveness itself was not altered. Indeed, β-adrenergic receptor density and affinity, cAMP levels, and PKA activity were comparable with wild-type controls (Fig. 7, A and B). In contrast, PP1 activity was selectively decreased, pointing towards a possible role of G{alpha}o* in its regulation. Decreased PP1 activity was found in close proximity to one of its targets (RyR2, Fig. 7D), which appears to be particularly sensitive to the balance between PKA and phosphatase regulation (7). The reduction in PP1 activity was significant, albeit modest (22%), and could be at least in part due to a decrease in the amount of PP1 that was present in the RyR2 complex in G{alpha}o*-expressing ventricles (Fig. 7E). The PP1 activity associated with RyR2 amounted to approximately one-half of the total phosphatase activity. This is in contrast to myocyte lysates in which PP2A activity predominated over PP1 activity (66 vs. 30% of total). The relative importance of select phosphatase isoforms to regulate target- and site-specific phosphorylation is not well known, but it is conceivable that the observed reduction in PP1 amount and/or activity in the RyR2 complex contribute to RyR2 hyperphosphorylation in G{alpha}o* mice.

Our findings support the concept of compartmentalized signaling domains in cardiac myocytes (12, 17, 43). In contrast to the RyR2, which is a very large protein (molecular mass of 565 kDa) that exists as a tetramer and can be immunoprecipitated when complexed with many associated proteins (3, 32), PLB is a rather small protein (molecular mass of 6.5 kDa) that assembles into a homopentamer (30). Whether or not localized changes in PP1 activity (or other signaling molecules) also exist in the vicinity of PLB in G{alpha}o* ventricles and potentially contribute to its hyperphosphorylation cannot be addressed with currently available reagents and techniques. Although we did not observe any global changes in cAMP/PKA signaling, spatially confined changes in response to G{alpha}o activation that might also contribute to the phenotype of G{alpha}o* mice cannot be ruled out.

Potential implications. In the present study, we used a gain-of-function approach to advance our understanding of G{alpha}o in the heart. It complements previous studies (23, 49), in which both isoforms of G{alpha}o were deleted. Using a loss-of function approach, we and others (49, 56) previously showed that coupling between the M2 receptor and Go is required for muscarinic inhibitory LCC regulation. We did not expect muscarinergic ICa-L regulation to be altered in transgenic G{alpha}o* mice, because constitutively active G{alpha} subunits are not prone to receptor activation: GTP triggers a conformational change in the G{alpha} "switch" regions that weakens the affinity between G{alpha} and Gβ{gamma} (11) and thereby precludes association with receptors. Indeed, carbachol diminished isoproterenol-induced ICa-L in both G{alpha}o*68 and wild types to the same extent (data not shown).

Observations made in the G{alpha}o* transgenic mice cannot be directly juxtaposed to findings from G{alpha}o knockout studies for two reasons: 1) in contrast to the transgenic model, deletion of G{alpha}o in the existing reports was not restricted to the heart, thereby allowing for potential systemic effects (particularly since Go is the most abundant G protein in the brain; Ref. 22); and 2) Gβ{gamma} expression was significantly reduced in G{alpha}o knockout mice (37, 49), so that no distinctions could be made whether phenotypic effects (such as the lack of inhibitory muscarinergic ICa-L regulation) were due to the loss of G{alpha}o or the concomitant reduction in Go β{gamma}-subunits. Nevertheless, the fact that transgenic G{alpha}o* expression in the present study did not lead to constitutive blunting of isoproterenol-induced ICa-L (as would be expected for constitutively active G{alpha}o* if the {alpha}-subunit of Go were the primary mediator) indirectly suggests that Go β{gamma}-subunits mediate muscarinic ICa-L regulation. This would be consistent with an inhibitory effect of Go β{gamma}-subunits on the opening probability of voltage-gated Ca2+ channels in neurons and neuroendocrine cells (13). The increase in ICa-L that we observed in the G{alpha}o* transgenic mice suggests that G{alpha}o may in fact exert an independent stimulatory effect on LCC. Opposing effects of G{alpha} and Gβ{gamma} subunits are not without precedent. For example, dual regulation of select adenylyl cyclase isoforms was shown for G{alpha}s and Gβ{gamma} (48). However, information about heterotrimeric G proteins subunit compositions as well as tissue distribution and isoform composition of their downstream targets is still limited. It is therefore not well understood whether counterregulatory effects by G{alpha} and Gβ{gamma} subunits that are released from the same heterotrimer do occur under physiological or pathophysiological conditions and what their functional consequences are.

The present study has one other potential implication: upregulation of pertussis toxin-sensitive G proteins from the Gi/o subfamily has long been recognized as a central feature of failing hearts in humans and a variety of animal models (53). It is generally believed that their upregulation in heart failure occurs in response to an increase in sympathetic drive and contributes to the blunting of contractile function (29). Reduced contraction upon G{alpha}i2 overexpression was reported in support of this notion (41). Since wild-type G{alpha}i2, which requires endogenous Gβ{gamma} to become activated, was used in that study, the relative contribution of G{alpha}i2 and β{gamma} subunits could not be discerned. While upregulation of G{alpha}i was immunochemically confirmed early on in many studies on the expression of pertussis toxin-sensitive G proteins in heart failure, G{alpha}o was often not examined, because its significance in regulating cardiac function was not appreciated at the time. Interestingly, G{alpha}o upregulation was recently shown to be among the top 20 predictors of heart failure development in different murine models (5). Since contractile function was markedly increased upon expression of constitutively active G{alpha}o*, the present study raises the possibility that G{alpha}o upregulation could potentially be a compensatory response in heart failure. The net effect that activation of Go protein will produce upon cellular receptor activation is difficult to predict, because it will represent the combined effects that its {alpha}- and β{gamma}-subunits produce upon dissociation (and those could possibly be counteractive, see above). Importantly, one also needs to consider concomitant Gi protein effects, because both Go and Gi proteins are closely related and tend to couple equally well to many receptors. It has been shown that targeted deletion of either G{alpha}o or G{alpha}i2 can lead to obliteration of the same functional effect despite the fact that the respective other subunits were normally expressed (10, 39, 49). Thus, both G proteins seem to be critical for mediating downstream receptor effects and may in fact to depend on each other, adding to the overall complexity of G protein signaling.

In conclusion, despite intense efforts for more than two decades, the functional role of Go protein has remained most elusive among G proteins in many tissues including the heart. The present study introduces a novel transgenic mouse model with cardiac-specific constitutive G{alpha}o activation. To our knowledge, it demonstrates for the first time marked stimulatory effects of constitutively active G{alpha}o* on cardiac Ca2+ cycling and contractile function, both in ventricular myocytes and in vivo. The molecular links between G{alpha}o* expression and the resulting phenotype remain to be elucidated but appear to involve (and are likely not limited to) alterations in PP1 activity. PP1 has garnered attention as a potential therapeutic target in heart failure (8). Further investigations of the relationship between G{alpha}o* and PP1 and the involvement of other signaling mediators in the healthy and diseased heart seem warranted and may lead to new opportunities for therapeutic design.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the National Heart Lung and Blood Institute Grant HL-072174 (to U. Mende).


   ACKNOWLEDGMENTS
 
We thank L. Du and A. Sharpe of the Transgenic Mouse Facility at Brigham and Women's Hospital for pronuclear injections and A. Marks and S. Reiken (Columbia University) for conducting RyR2-related experiments. H. Shin provided expert technical assistance in preparing histological specimens that were evaluated by F. J. Schoen (Brigham and Women's Hospital).


   FOOTNOTES
 

Address for reprint requests and other correspondence: U. Mende, Cardiovascular Research Center, Division of Cardiology, Rhode Island Hospital, Coro Center, 5thfloor, Rm. 5105, 1 Hoppin St., Providence, RI 02903 (e-mail: Ulrike_Mende{at}brown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akhter SA, Skaer CA, Kypson AP, McDonald PH, Peppel KC, Glower DD, Lefkowitz RJ, Koch WJ. Restoration of β-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer. Proc Natl Acad Sci USA 94: 12100–12105, 1997.[Abstract/Free Full Text]
  2. Bers DM. Cardiac ryanodine receptor phosphorylation: target sites and functional consequences. Biochem J 396: E1–3, 2006.[CrossRef][Medline]
  3. Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol 37: 417–429, 2004.[CrossRef][Web of Science][Medline]
  4. Bers DM, Eisner DA, Valdivia HH. Sarcoplasmic reticulum Ca2+ and heart failure: roles of diastolic leak and Ca2+ transport. Circ Res 93: 487–490, 2003.[Free Full Text]
  5. Blaxall BC, Spang R, Rockman HA, Koch WJ. Differential myocardial gene expression in the development and rescue of murine heart failure. Physiol Genomics 15: 105–114, 2003.[Abstract/Free Full Text]
  6. Braz JC, Gregory K, Pathak A, Zhao W, Sahin B, Klevitsky R, Kimball TF, Lorenz JN, Nairn AC, Liggett SB, Bodi I, Wang S, Schwartz A, Lakatta EG, DePaoli-Roach AA, Robbins J, Hewett TE, Bibb JA, Westfall MV, Kranias EG, Molkentin JD. PKC-{alpha} regulates cardiac contractility and propensity toward heart failure. Nat Med 10: 248–254, 2004.[CrossRef][Web of Science][Medline]
  7. Carter S, Colyer J, Sitsapesan R. Maximum phosphorylation of the cardiac ryanodine receptor at serine-2809 by protein kinase a produces unique modifications to channel gating and conductance not observed at lower levels of phosphorylation. Circ Res 98: 1506–1513, 2006.[Abstract/Free Full Text]
  8. Champion HC. Targeting protein phosphatase 1 in heart failure. Circ Res 96: 708–710, 2005.[Free Full Text]
  9. Chen CA, Manning DR. Regulation of G proteins by covalent modification. Oncogene 20: 1643–1652, 2001.[CrossRef][Web of Science][Medline]
  10. Chen F, Spicher K, Jiang M, Birnbaumer L, Wetzel GT. Lack of muscarinic regulation of Ca2+ channels in Gi2{alpha} gene knockout mouse hearts. Am J Physiol Heart Circ Physiol 280: H1989–H1995, 2001.[Abstract/Free Full Text]
  11. Clapham DE. The G-protein nanomachine. Nature 379: 297–299, 1996.[CrossRef][Medline]
  12. Dodge-Kafka KL, Langeberg L, Scott JD. Compartmentation of cyclic nucleotide signaling in the heart: the role of A-kinase anchoring proteins. Circ Res 98: 993–1001, 2006.[Abstract/Free Full Text]
  13. Dolphin AC. G protein modulation of voltage-gated calcium channels. Pharmacol Rev 55: 607–627, 2003.[Abstract/Free Full Text]
  14. Ellingsen O, Davidoff AJ, Prasad SK, Berger HJ, Springhorn JP, Marsh JD, Kelly RA, Smith TW. Adult rat ventricular myocytes cultured in defined medium: phenotype and electromechanical function. Am J Physiol Heart Circ Physiol 265: H747–H754, 1993.[Abstract/Free Full Text]
  15. Eschenhagen T, Laufs U, Schmitz W, Scholz H, Warnholtz A, Weil J, Schafer HJ. Enrichment of G protein {alpha}-subunit mRNAS in the AV-conducting system of the mammalian heart. J Mol Cell Cardiol 27: 2249–2263, 1995.[CrossRef][Web of Science][Medline]
  16. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 157: 378–417, 1988.[Web of Science][Medline]
  17. Fischmeister R, Castro LR, Abi-Gerges A, Rochais F, Jurevicius J, Leroy J, Vandecasteele G. Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases. Circ Res 99: 816–828, 2006.[Abstract/Free Full Text]
  18. Gehrmann J, Meister M, Maguire CT, Martins DC, Hammer PE, Neer EJ, Berul CI, Mende U. Impaired parasympathetic heart rate control in mice with a reduction of functional G protein β{gamma} subunits. Am J Physiol Heart Circ Physiol 282: H445–H456, 2002.[Abstract/Free Full Text]
  19. Greif GJ, Sodickson DL, Bean BP, Neer EJ, Mende U. Altered regulation of potassium and calcium channels by GABAB and adenosine receptors in hippocampal neurons from mice lacking G{alpha}o. J Neurophysiol 83: 1010–1018, 2000.[Abstract/Free Full Text]
  20. Gudermann T, Kalkbrenner F, Schultz G. Diversity and selectivity of receptor-G protein interaction. Annu Rev Pharmacol Toxicol 36: 429–459, 1996.[Web of Science][Medline]
  21. Hagemann D, Xiao RP. Dual site phospholamban phosphorylation and its physiological relevance in the heart. Trends Cardiovasc Med 12: 51–56, 2002.[CrossRef][Web of Science][Medline]
  22. Huff RM, Axton JM, Neer EJ. Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex. J Biol Chem 260: 10864–10871, 1985.[Abstract/Free Full Text]
  23. Jiang M, Gold MS, Boulay G, Spicher K, Peyton M, Brabet P, Srinivasan Y, Rudolph U, Ellison G, Birnbaumer L. Multiple neurological abnormalities in mice deficient in the G protein Go. Proc Natl Acad Sci USA 95: 3269–3274, 1998.[Abstract/Free Full Text]
  24. Kamp TJ, Hell JW. Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ Res 87: 1095–1102, 2000.[Abstract/Free Full Text]
  25. Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, Lefkowitz RJ. Cardiac function in mice overexpressing the β-adrenergic receptor kinase or a βARK inhibitor. Science 268: 1350–1353, 1995.[Abstract/Free Full Text]
  26. Lehnart S, Marks AR. Regulation of ryanodine receptors in the heart. Circ Res 101: 746–749, 2007.[Free Full Text]
  27. Lim CC, Helmes MH, Sawyer DB, Jain M, Liao R. High-throughput assessment of calcium sensitivity in skinned cardiac myocytes. Am J Physiol Heart Circ Physiol 281: H969–H974, 2001.[Abstract/Free Full Text]
  28. Liu GX, Zhou J, Nattel S, Koren G. Single-channel recordings of a rapid delayed rectifier current in adult mouse ventricular myocytes: basic properties and effects of divalent cations. J Physiol 556: 401–413, 2004.[Abstract/Free Full Text]
  29. Lohse MJ, Engelhardt S, Eschenhagen T. What is the role of β-adrenergic signaling in heart failure? Circ Res 93: 896–906, 2003.[Abstract/Free Full Text]
  30. MacLennan DH, Kranias EG. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol 4: 566–577, 2003.[CrossRef][Web of Science][Medline]
  31. Marks AR. A guide for the perplexed: towards an understanding of the molecular basis of heart failure. Circulation 107: 1456–1459, 2003.[Free Full Text]
  32. Marx S. Ion channel macromolecular complexes in the heart. J Mol Cell Cardiol 35: 37–44, 2003.[CrossRef][Web of Science][Medline]
  33. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101: 365–376, 2000.[CrossRef][Web of Science][Medline]
  34. McIntire WE, Dingus J, Schey KL, Hildebrandt JD. Characterization of the major bovine brain Go alpha isoforms. Mapping the structural differences between the alpha subunit isoforms identifies a variable region of the protein involved in receptor interactions. J Biol Chem 273: 33135–33141, 1998.[Abstract/Free Full Text]
  35. Mende U, Kagen A, Cohen A, Aramburu J, Schoen FJ, Neer EJ. Transient cardiac expression of constitutively active G{alpha}q leads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways. Proc Natl Acad Sci USA 95: 13893–13898, 1998.[Abstract/Free Full Text]
  36. Mende U, Kagen A, Meister M, Neer EJ. Signal transduction in atria and ventricles of mice with transient cardiac expression of activated G protein {alpha}q. Circ Res 85: 1085–1091, 1999.[Abstract/Free Full Text]
  37. Mende U, Zagrovic B, Cohen A, Li Y, Valenzuela D, Fishman MC, Neer EJ. Effect of deletion of the major brain G-protein {alpha} subunit ({alpha}o) on coordination of G-protein subunits and on adenylyl cyclase activity. J Neurosci Res 54: 263–272, 1998.[CrossRef][Web of Science][Medline]
  38. Mumby SM, Heukeroth RO, Gordon JI, Gilman AG. G-protein {alpha}-subunit expression, myristoylation, and membrane association in COS cells. Proc Natl Acad Sci USA 87: 728–732, 1990.[Abstract/Free Full Text]
  39. Nagata K, Ye C, Jain M, Milstone DS, Liao R, Mortensen RM. G{alpha}i2 but not G{alpha}i3 is required for muscarinic inhibition of contractility and calcium currents in adult cardiomyocytes. Circ Res 87: 903–909, 2000.[Abstract/Free Full Text]
  40. Offermanns S. G-proteins as transducers in transmembrane signalling. Prog Biophys Mol Biol 83: 101–130, 2003.[CrossRef][Web of Science][Medline]
  41. Rau T, Nose M, Remmers U, Weil J, Weissmuller A, Davia K, Harding S, Peppel K, Koch WJ, Eschenhagen T. Overexpression of wild-type G{alpha}i-2 suppresses β-adrenergic signaling in cardiac myocytes. FASEB J 17: 523–525, 2003.[Abstract/Free Full Text]
  42. Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. Nature 415: 206–212, 2002.[CrossRef][Medline]
  43. Steinberg SF, Brunton LL. Compartmentation of G protein-coupled signaling pathways in cardiac myocytes. Ann Rev Pharmacol Toxicol 41: 751–773, 2001.[CrossRef][Web of Science][Medline]
  44. Strathmann M, Wilkie TM, Simon MI. Alternative splicing produces transcripts encoding two forms of the {alpha} subunit of GTP-binding protein Go. Proc Natl Acad Sci USA 87: 6477–6481, 1990.[Abstract/Free Full Text]
  45. Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J. Tissue-specific regulation of the {alpha}-myosin heavy chain gene promoter in transgenic mice. J Biol Chem 266: 24613–24620, 1991.[Abstract/Free Full Text]
  46. Tanaka M, Treloar H, Kalb RG, Greer CA, Strittmatter SM. Go protein-dependent survival of primary accessory olfactory neurons. Proc Natl Acad Sci USA 96: 14106–14111, 1999.[Abstract/Free Full Text]
  47. Taussig R, Iniguez-Lluhi JA, Gilman AG. Inhibition of adenylyl cyclase by Gi alpha. Science 261: 218–221, 1993.[Abstract/Free Full Text]
  48. Taussig R, Quarmby LM, Gilman AG. Regulation of purified type I and type II adenylylcyclases by G protein β{gamma} subunits. J Biol Chem 268: 9–12, 1993.[Abstract/Free Full Text]
  49. Valenzuela D, Han X, Mende U, Fankhauser C, Mashimo H, Huang P, Pfeffer J, Neer EJ, Fishman MC. G{alpha}o is necessary for muscarinic regulation of Ca2+ channels in mouse heart. Proc Natl Acad Sci USA 94: 1727–1732, 1997.[Abstract/Free Full Text]
  50. Wang Y, Windh RT, Chen CA, Manning DR. N-myristoylation and β{gamma} play roles beyond anchorage in the palmitoylation of the G protein {alpha}o subunit. J Biol Chem 274: 37435–37442, 1999.[Abstract/Free Full Text]
  51. Wedegaertner PB, Wilson PT, Bourne HR. Lipid modifications of trimeric G proteins. J Biol Chem 270: 503–506, 1995.[Free Full Text]
  52. Wehrens XH, Lehnart SE, Marks AR. Intracellular calcium release and cardiac disease. Annu Rev Physiol 67: 69–98, 2005.[CrossRef][Web of Science][Medline]
  53. Wettschureck N, Offermanns S. Mammalian G proteins and their cell type specific functions. Physiol Rev 85: 1159–1204, 2005.[Abstract/Free Full Text]
  54. Wichelhaus A, Russ M, Petersen S, Eckel J. G protein expression and adenylate cyclase regulation in ventricular cardiomyocytes from STZ-diabetic rats. Am J Physiol Heart Circ Physiol 267: H548–H555, 1994.[Abstract/Free Full Text]
  55. Wolf WP, Spicher K, Haase H, Schulze W. Immunocytochemical localization of the G-protein subunit, Go{alpha}, in rat heart. Implications for a role of Go{alpha} in secretion of cardiac hormones. J Mol Cell Cardiol 30: 1149–1162, 1998.[CrossRef][Web of Science][Medline]
  56. Ye C, Sowell MO, Vassilev PM, Milstone DS, Mortensen RM. G{alpha}i2, G{alpha}i3 and G{alpha}o are all required for normal muscarinic inhibition of the cardiac calcium channels in nodal/atrial-like cultured cardiocytes. J Mol Cell Cardiol 31: 1771–1781, 1999.[CrossRef][Web of Science][Medline]
  57. Zubiaur M, Neer EJ. Nerve growth factor changes G protein levels and localization in PC12 cells. J Neurosci Res 35: 207–217, 1993.[CrossRef][Web of Science][Medline]



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