Adenoviral Gene Transfer of the Human V2 Vasopressin Receptor Improves Contractile Force of Rat Cardiomyocytes Karl-Ludwig Laugwitz, Martin Ungerer, Torsten Schöneberg, Hans-Jörg Weig, Kai Kronsbein, Alessandra Moretti, Katrin Hoffmann, Melchior Seyfarth, Günter Schultz and Albert Schömig Circulation. 1999;99:925-933 doi: 10.1161/01.CIR.99.7.925 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1999 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539

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Adenoviral Gene Transfer of the Human V2 Vasopressin Receptor Improves Contractile Force of Rat Cardiomyocytes Karl-Ludwig Laugwitz,* MD; Martin Ungerer,* MD; Torsten Scho¨neberg, MD; Hans-Jo¨rg Weig, MD; Kai Kronsbein, BS; Alessandra Moretti, PhD; Katrin Hoffmann, MS; Melchior Seyfarth, MD; Gu¨nter Schultz, MD; Albert Scho¨mig, MD Background—In congestive heart failure, high systemic levels of the hormone arginine vasopressin (AVP) result in vasoconstriction and reduced cardiac contractility. These effects are mediated by the V1 vasopressin receptor (V1R) coupled to phospholipase C b-isoforms. The V2 vasopressin receptor (V2R), which promotes activation of the Gs/adenylyl cyclase system, is physiologically expressed in the kidney but not in the myocardium. Expression of a recombinant V2R (rV2R) in the myocardium could result in a positive inotropic effect via the endogenous high concentrations of AVP in heart failure. Methods and Results—A recombinant adenovirus encoding the human V2R (Ad-V2R) was tested for its ability to modulate the cardiac Gs/adenylyl cyclase system and to potentiate contractile force in rat ventricular cardiomyocytes and in H9c2 cardiomyoblasts. Ad-V2R infection resulted in a virus concentration-dependent expression of the transgene and led to a marked increase in cAMP formation in rV2R-expressing cardiomyocytes after exposure to AVP. Single-cell shortening measurements showed a significant agonist-induced contraction amplitude enhancement, which was blocked by the V2R antagonist, SR 121463A. Pretreatment of Ad-V2R-infected cardiomyocytes with AVP led to desensitization of the rV2R after short-term agonist exposure but did not lead to further loss of receptor function or density after long-term agonist incubation, thus demonstrating resistance of the rV2R to downregulation. Conclusions—Adenoviral gene transfer of the V2R in cardiomyocytes can modulate the endogenous adenylyl cyclasesignal transduction cascade and can potentiate contraction amplitude in cardiomyocytes. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in congestive heart failure by bypassing the desensitized b-adrenergic receptor signaling. (Circulation. 1999;99:925-933.) Key Words: receptors n myocardial contraction n heart failure

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any neurotransmitters, hormones, and autocrine and paracrine factors exert their physiological functions by binding to heptahelical receptors which activate or inhibit specific signal transduction pathways that couple with different classes of heterotrimeric G proteins.1,2 Vasopressin receptors, belonging to the superfamily of heptahelical receptors, exist as 4 subtypes: the V1A vasopressin receptor (V1AR), which is mainly expressed in the endothelium, smooth muscle cells, and the myocardium; the V1BR and the probably identical V3R subtype, which is expressed in the hypophysis; and the V2 vasopressin receptor (V2R), which is exclusively expressed in the renal collecting tubule cells. Stimulation of V2R results in the activation of the Gs/adenylyl cyclase system and promotes the reabsorption of fluid via insertion of water pores into the luminal membrane.3,4 Congestive heart failure is a syndrome that confers significant morbidity and mortality despite recent advances in

clinical therapy.5 Numerous compensation mechanisms occur in this syndrome, including an increased activity of the sympathoadrenergic system and of the renin-angiotensinaldosteron complex and an increased release of several peptide hormones, such as arginine vasopressin (AVP) and atrial natriuretic peptide. The elevated concentrations of catecholamines and AVP in the systemic circulation of patients with congestive heart failure correlate with their prognosis.6,7 Despite high levels of catecholamines, the failing myocardium is not able to build up a sufficient blood supply for the organism. In the failing heart, the b-adrenergic signaling cascade is functionally inactivated by a selective downregulation of b1-adrenergic receptor (AR)8 and an upregulation of the b-AR kinase, an enzyme that specifically phosphorylates and uncouples the activated b-AR.9 The present study investigates the feasibility of overexpressing a heterologous cAMP-coupling receptor in cardio-

Received May 15, 1998; revision received September 10, 1998; accepted October 5, 1998. *Dr Laugwitz and Dr Ungerer contributed equally to this work. From I. Medizinische Klinik, Klinikum rechts der Isar, and Deutsches Herzzentrum Mu¨nchen, Germany; and Institut fu¨r Pharmakologie (T.S., G.S.), Freie Universita¨t Berlin, Germany. Correspondence to Karl-Ludwig Laugwitz, MD, I. Medizinische Klinik, Klinikum rechts der Isar, Ismaninger Straße 22, D-81675 Mu¨nchen, Germany. E-mail [email protected] © 1999 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

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myocytes which could potentially bypass the defective b-adrenergic signal transduction observed in heart failure. We chose as model systems, adult ventricular cardiomyocytes and cardiomyoblasts. H9c2 cardiomyoblasts were used because they possess b-adrenergic signal transduction characteristics similar to the human myocardium and they can be cultured in a standardized manner, thereby facilitating the comparability of large-scale experiments. Isolated rat adult ventricular cardiomyocytes retain most contractile properties of the intact adult heart. Recently, replication-deficient recombinant adenoviral vectors have been used for efficient gene transfer into the myocardium and into isolated cardiac myocytes.10,11 Adenoviral vectors have important characteristics that make them well suited for myocardial gene transfer. The efficiency and expression levels of adenovirus-mediated gene transfer in vitro and in vivo are substantially better than those seen with other types of gene-delivery systems, such as liposomal transfection or direct plasmid DNA injection.10 The aim of this study was to explore whether an adenovirally mediated transfer of the human V2R would lead to a sufficient recruitment of the endogenous Gs/ adenylyl cyclase system to potentiate contraction amplitude in cardiomyocytes.

Methods Construction and Purification of Recombinant Adenovirus Generation of a recombinant (E1 deficient) adenovirus (Serotype 5) carrying the human V2R has been described recently.12 Briefly, the coding sequence of the hemagglutinin-tagged wild-type V2R, including the nontissue-specific cytomegalovirus promoter and SV40 polyadenylation signal, was taken from the hemagglutinin-V2pcD-PS vector12 and inserted into the polylinker sequence of the pDE1sp1B vector.13 The resulting plasmid was then cotransfected with the pJM17 plasmid14 into subconfluent HEK 293 cells by using a modified calcium phosphate coprecipitation method.15 After plaque isolation, recombinant virus, referred to as Ad-V2R, was amplified, and individual virus stocks were analyzed by polymerase chain reaction and restriction analysis. After isolation, Ad-V2R were prepared at large scale as described previously.12 Adenoviral titers were determined using plaque titration on HEK 293 cells.15

Cell Culture and Adenoviral Infection of H9c2 Cardiomyoblasts H9c2 cardiomyoblasts (ATCC CRL 1446, cardiac myoblasts from rat) were cultured in monolayers in DMEM, 10% fetal bovine serum, 2 mmol/L glutamine, penicillin (100 IU/mL), and streptomycin (100 mg/mL) in 7% CO2 in a humidified incubator at 37°C. The cells were used for the individual experiments 36 to 48 hours after adenoviral infection (performed as described).12

Preparation and Culture of Adult Ventricular Cardiomyocytes Single calcium-tolerant ventricular myocytes were isolated from 12to 16-week-old male Wistar rats and cultured in M199 medium (supplemented with vitamins, nonessential amino acids, 25 mmol/L HEPES, 10 mg/mL insulin, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 100 mg/mL gentamicin), on laminin-precoated dishes (5- to 10 mg/cm2 at a density of 105 cells/cm2), in a humidified atmosphere (5% CO2) at 37°C. The infection of the ventricular cardiomyocytes with the adenoviruses was performed 6 to 8 hours after plating in M199 culture medium.

Confocal Laser Scanning Microscopy Immunofluorescence microscopy of Ad-V2R-infected cardiomyoblasts was performed as described previously.12

Contraction Experiments Measurement of contraction amplitude of Ad-V2R- or Ad-bgalactosidase (Gal)-infected rat cardiomyocytes was performed with an electro-optical monitoring system.16 The contraction amplitude of the cardiomyocytes was recorded because excellent correlation between this parameter and contraction velocity was reported for an identical preparation of adult cardiomyocytes.17 The experiments were performed on ventricular cardiomyocytes in a single-cell investigation system (Scientific Instruments), in a temperature-controlled cuvette (37°C) at a constant medium flow of 0.5 mL/min and at a constant electrical field. As medium, a 1.8-mmol/L Ca21-Tyrode’s-solution was used. The cardiomyocytes were paced by an external stimulation of 50 V and an 800-ms pulse duration to achieve a contraction frequency of '70 minutes21. After the contraction amplitude reached stability, the experiments were started by applying increasing concentrations of AVP or isoproterenol. The AVP-treated cells were finally superfused with isoproterenol (0.1 mmol/L) to control comparability.

Determination of Intracellular cAMP Concentrations and Sarcolemmal Adenylyl Cyclase Activity These assays were performed in ventricular cardiomyocytes or H9c2 cardiomyoblasts plated at the density of 13105 cells/cm2, following the protocols previously described.18,19

Radioligand Binding Cells were harvested 48 hours after adenoviral infection. Membranes were prepared and radioligand binding was performed as described.12 The protein content of each sample was determined by the method of Bradford.20

b-Galactosidase Expression To estimate infection efficiencies, cells were infected with varying concentrations of a recombinant adenovirus carrying the lacZ gene under the expression control of a Rous sarcoma virus promoter. After adenoviral infection, the cells were fixed in 0.05% glutaraldehyde in PBS for 10 minutes at 4°C and then stained with 3 mmol/L K4Fe(CN)6, 3 mmol/L K3Fe(CN)6, 1 mmol/L MgCl2, 15 mmol/L NaCl, and 1 mg/mL X-Gal-(5-bromo-4-chloro-3-indolyl-b-dgalactopyranoside) in 44 mmol/L HEPES for 1 hour at 37°C.

Receptor Desensitization and Downregulation Protocol Desensitization of b-adrenergic or V2R was induced by incubating the cells for 15 minutes at 37°C in PBS containing 1 mmol/L of isoproterenol or 10 nmol/L of vasopressin and 1 mmol/L of IBMX. For receptor downregulation, the cells were treated for 20 hours with the indicated agonists. The incubation was stopped by washing the cells 3 times in PBS. Afterward, the intracellular cAMP content or the density of rV2R or b-AR in the cell membranes was determined.

Data Analysis To compare the statistical significance of the differences between the means of 2 independent groups, the Student t test with 2-tailed distribution was used. The data presented in Figures 2, 3 and 5 were analyzed by 1-way ANOVA followed by Scheffe´’s analysis.

Results Infection With Ad-bGal and Ad-V2R Adenoviral constructs for the human V2R (Ad-V2R) and LacZ (Ad-bGal) were used in all experiments. Robust expression of the 2 adenoviral transgenes was demonstrated after infection of both cardiomyoblasts and isolated adult cardiomyocytes. Figure

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Figure 1. Cytochemical and immunofluorescence studies in H9c2 cardiomyoblasts infected with recombinant adenoviruses. A, Ad-bGal-infected cardiomyoblasts (moi: 100 pfu/cell, right) were compared with Ad-V2R-infected control cells (moi: 100 pfu/cell, left). After 48 hours, X-Gal staining was performed as described in Methods. B, After infection with Ad-V2R (moi: 30 pfu/cell, right) or with Ad-bGal (moi: 30 pfu/cell, left), rV2R-expressing cardiomyoblasts were probed with an affinitypurified polyclonal antibody (10 mg/mL) directed against the C-terminal 29 amino acids of the human V2 receptor. Experiments were performed in permeabilized cells. Fluorescence images were obtained with a confocal laser scanning microscope (MRC-600, BioRad), using FITC-linked anti-rabbit IgG specific secondary antibodies. Figure is representative of 3 independent experiments.

1A shows that after infection with the adenovirus for b-Gal (moi: 100 pfu/cell) and subsequent staining with X-Gal, almost all H9c2 cardiomyoblasts showed a positive nuclear staining, whereas Ad-V2R-infected cardiomyoblasts remained colorless under these conditions (Figure 1A). Expression of the adenoviral transgene for the V2R was demonstrated by confocal fluorescence microscopy using a polyclonal antibody raised against a C-terminal peptide of the receptor. As shown in Figure 1B, '80% of the Ad-V2Rinfected H9c2 cardiomyoblasts showed an intense, intracellular staining of the rV2R transgene after permeabilization. Radioligand binding with 3H-AVP documented marked expression of rV2R after infection with Ad-V2R. A concentration-dependent effect was observed with increasing titers of the adenoviral construct for the V2R which resulted in a Bmax value for 3H-AVP-binding of 9986119 fmol/mg protein at moi of 100 pfu/cell (Figure 2A). The decrease in rV2R density at higher adenoviral titers is probably a result of viral toxicity. Noninfected control cardiomyoblast membranes showed specific 3H-AVP binding with a Bmax-value of 270687 fmol/mg protein resulting from endogenously ex-

pressed V1AR. Infection with either Ad-bGal or Ad-V2R did not significantly alter b-AR density in the membranes of H9c2 cardiomyoblasts (81618 fmol/mg protein). The Kd value for 3H-AVP binding was 0.1660.09 nmol/L. To differentiate the receptor subtype populations, the selective V2R antagonist SR 121463A, which binds to V2R (in comparison with V1R) with a selectivity of 2400:1, was used.21 Figure 2B shows that in cardiomyoblasts infected with either Ad-bGal or Ad-V2R (moi: 100 pfu/cell), a native population of V1AR existed with a Bmax-value of '270 fmol/mg protein. In Ad-V2Rinfected cardiomyoblasts, the total amount of binding sites for 3 H-AVP increased to 8806110 fmol/mg protein. By application of the selective V2R antagonist SR 121463A, this increase in 3 H-AVP binding was almost completely blocked (Figure 2B). These data demonstrate the dominant expression of the rV2 receptor in comparison with the endogenous V1A population in Ad-V2R-infected cells. Also, in adult ventricular cardiomyocytes, we detected a 3-fold increase in 3H-AVP binding after infection with Ad-V2R (Bmax value of 155618 fmol/mg protein versus 4869 fmol/mg protein in Ad-bGal-infected control cardiomyocytes [n54];

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Figure 2. A, Determination of V2R expression levels in Ad-V2R-infected cardiomyoblasts. H9c2 cardiomyoblasts were infected with increasing amounts of Ad-V2R (moi: 10 to 1000 pfu/cell). For determination of Bmax values, prepared membranes were incubated with 3H-AVP at concentrations ranging from 0.2 to 8 nmol/L in the presence or absence of unlabelled AVP (5 mmol/L). The data represent the mean6SEM of 3 independent experiments, each performed in triplicate. B, Characterization of the specific 3 H-AVP binding sites in Ad-V2R- and bGal-infected cardiomyoblasts. H9c2 cardiomyoblasts were either infected with Ad-bGal (moi: 100 pfu/cell, left) or with Ad-V2R (moi: 100 pfu/cell, right). Radioligand binding assays were performed in the absence (shaded bars) or presence (solid bars) of 0.1 mmol/L of the selective V2R antagonist SR 121463A. The data represent the mean6SEM of 3 independent experiments, each performed in triplicate. **P,0.0015.

moi: 100 pfu/cell). The basal and heterologous expression levels of V1R and V2R were each 6-fold lower in cardiomyocytes versus H9c2 cardiomyoblasts. To examine the time dependence and the optimum adenoviral titer for rV2R expression in cardiomyoblasts, receptor-dependent cAMP formation was determined in intact cardiomyoblasts infected with increasing titers of Ad-V2R. A concentration-dependent effect was seen with a maximum 4-fold increase at an moi of 100 pfu Ad-V2R/ cell, corresponding to 3H-AVP binding experiments in Figure 2A. Intracellular cAMP formation peaked at 48 to 72 hours after infection at optimal adenoviral titer.

Intracellular cAMP Formation and Sarcolemmal Adenylyl Cyclase Activity After Infection With Ad-V2R

In Ad-bGal-infected or noninfected adult rat ventricular cardiomyocytes, intracellular cAMP formation did not change after addition of increasing concentrations of AVP.

After V2R transgene expression, receptor-stimulated intracellular cAMP production reached a maximum increase of 4-fold with an EC50 value of '0.5 nmol/L in ventricular cardiomyocytes (Figure 5A). Sarcolemmal adenylyl cyclase activity was measured in H9c2 cardiomyoblasts. In membrane fractions of Ad-V2R-infected cardiomyoblasts, AVP-stimulated membrane adenylyl cyclase activity increased to values 35-fold above basal. The EC50 value for the AVP-stimulated adenylyl cyclase activity was calculated to be 0.16 nmol/L. In comparison, a maximum 7-fold increase in cyclase activity was observed after stimulation of endogenous b-ARs with up to 10 mmol/L of isoproterenol. The half-maximal concentration for isoproterenol-stimulated cAMP formation was observed at 20 nmol/L (data not shown).

Homologous and Heterologous Receptor Desensitization and Downregulation of rV2R in Comparison to Native b-AR Receptor regulation of the heterologous protein was studied (1) after short-term (15 minutes) agonist exposure corre-

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Figure 4A and 4B show the functional effect of homologous desensitization on cAMP formation in Ad-V2R-infected cardiomyoblasts. Control concentration-response curves of the Ad-V2R-infected cardiomyoblasts showed EC50 values of '0.15 and 12 nmol/L for AVP and isoproterenol, respectively. The homologous receptor desensitization resulted in a reduction of maximal cAMP stimulation of 18% for the rV2R and of 42% for the b-AR. Additionally, the agonist concentration needed for half-maximal receptor stimulation shifted to higher EC50 values (from 0.15 to 1.2 nmol/L for AVP and from 15 to 50 nmol/L for isoproterenol). Figure 4C and 4D demonstrate the effect of long-term agonist incubation for a time which is commonly sufficient to induce complete homologous receptor downregulation. In Ad-V2R-infected cells, maximal receptor-dependent cAMP production via rV2R was only reduced by '12%, whereas cAMP formation after stimulation of endogenous b-AR was almost blunted (78% reduction). Figure 5A shows the effect of Ad-V2R infection on AVPinduced cAMP generation in adult ventricular myocytes. Also, in these cells, the effect of short-term and long-term agonist incubation was tested (Figure 5B). It can be seen that in cardiomyocytes, 15 minutes of agonist exposure decreased AVP-dependent cAMP formation by '20%, whereas no further decrease after long-term incubation with AVP occurred (Figure 5B). This is in contrast to the marked loss of adenlyl cyclase b-adrenergic stimulation observed in rat ventricular cardiomyocytes after long-term incubation with isoproterenol.

Physiological Effect of rV2R Expression on Contraction Amplitude in Rat Ventricular Cardiomyocytes Figure 3. Studies of ligand-induced receptor downregulation. Radioligand binding experiments with 3H-AVP and 3H-CGP 12177 were performed on crude membrane extracts from Ad-V2R-infected cardiomyoblasts (A) and ventricular cardiomyocytes (B). Cells were infected with Ad-V2R at a moi of 80 pfu/cell and b-AR- and V2R-densities were determined after agonist exposure of isoproterenol (solid bars) and AVP (shaded bars) for 0 and 15 minutes and 20 hours. The data represent the mean6SEM of 3 independent experiments, each performed in triplicate. *P,0.05; **P,0.0005.

sponding to the time frame in which receptor desensitization by phosphorylation occurs, and (2) after long-term (20 hours) agonist exposure, which is generally considered to simulate complete receptor downregulation and to correspond well to the long-term downregulation of b-AR seen in heart failure. Radioligand binding experiments were used to compare the regulation of the rV2R with that of the endogenous b-AR in cardiomyoblasts and adult ventricular cardiomyocytes (Figure 3A1B). Ad-V2R-infected cells were pretreated with either AVP or isoproterenol at concentrations of 10 nmol/L and 1 mmol/L, respectively. After 20 hours of exposure to isoproterenol, b-AR density was significantly downregulated, whereas V2R density remained stable after 20 hours of incubation with AVP (Figure 3A, P,0.05 and 3B, P,0.0005). No heterologous receptor regulation was found, as determined by measuring V2R density after isoproterenol or of b-AR density after AVP stimulation (data not shown).

To study the effect of heterologous expression of the rV2R on contractile responsiveness of ventricular cardiomyocytes, myocyte shortening was measured after infection with an moi of 100 pfu/cell of Ad-V2R. Cell culture conditions were chosen to maintain stable contraction characteristics of cardiomyocytes. Rat cardiomyocytes were electrically stimulated at a rate of '70 contractions per minute (Figure 6A and 6B). The baseline contraction amplitude was in the range of 3.4 to 3.8 mm and was not significantly altered by adenoviral infection. Baseline and isoproterenol-dependent contraction amplitude did not differ significantly between freshly isolated cardiomyocytes and isolated cardiomyocytes after 48 to 72 hours of culture (data not shown), which corresponded well to data from identical cell preparations.22 Superfusion of AdV2R-infected cardiomyocytes with AVP led to a significant increase in the contraction amplitude to the same extent as could be reached by b-adrenergic stimulation (Figure 6A). Control cells infected with Ad-bGal did not show any contractile response to stimulation with AVP at the submillimolar concentrations used (Figure 6B). The effect of AVP in Ad-V2R-infected cells was concentration-dependent and reached a maximum 3-fold increase in contraction amplitude. The observed EC50 was in the subnanomolar range, well below concentrations needed for stimulation of the natively present V1AR. Addition of the V2 antagonist, SR 121463A, inhibited the AVP-induced increase in contraction amplitude by 65% (inset of Figure 6A).

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Figure 4. Homologous desensitization studies for rV2R and endogenous b-AR in cardiomyoblasts. Ad-V2R-infected cardiomyoblasts (moi: 80 pfu/cell) were homologously desensitized for 15 minutes (A and B) or homologously downregulated for 20 hours (C and D) by preincubation with either AVP or isoproterenol. After washing, the cells were stimulated with increasing concentrations of the same agonists. Intracellular cAMP accumulation was determined and is shown as fold increase over basal values. The data represent mean6SEM of 3 independent experiments.

Discussion In the present study, we demonstrate that a heterologous receptor is capable of modulating cardiac adenylyl cyclasesignal transduction and is able to improve contractile function of cardiomyocytes.

Adenoviral Gene Transfer to Cardiomyocytes Adenoviral infection of cultured cardiomyoblasts or isolated cardiomyocytes resulted in an almost 100% transduction efficiency as assessed by X-Gal staining. Also, we

have demonstrated in immunofluorescence studies and radioligand binding that infection with Ad-V2R led to a robust transgene expression. Adenoviral infection did not enhance cAMP formation, per se, because there was no difference in the amount of AVP-stimulated cAMP formation between uninfected myocytes and those infected with high titers of Ad-bGal. In immunoblotting experiments, Ad-V2R infection did not significantly affect levels of G protein a-subunits of the Gs- and Gi-family, as assessed by others.23 In conclusion,

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Figure 5. A, cAMP formation in adult ventricular cardiomyocytes infected with Ad-V2R or Ad-bGal at a moi of 100 pfu/cell and stimulated with increasing concentrations of AVP (10 pmol/L to 0.1 mmol/L). B, Bar diagrams of cAMP formation after short-term incubation with AVP 10 nmol/L (15 minutes), demonstrating homologous desensitization; or long-term incubation for 20 hours, demonstrating the absence of any further downregulation of rV2R. cAMP accumulation is shown as fold increase over basal values. Data represent mean values6SEM of 4 independent experiments. *P,0.01 versus basal value.

adenovirus-mediated gene transfer proved to be an efficient and feasible gene delivery system to cardiac cells with our vectors in vitro.

Overexpression of the rV2R and Functional Implications in Cardiomyocytes After adenoviral infection, rV2R signaling was studied by measuring the accumulation of intracellular cAMP in intact myocytes or by studying the activation of adenylyl cyclase in sarcolemmal membranes. AVP-stimulated adenylyl cyclase activity increased to a maximum of 35-fold over basal in infected cardiomyoblasts. In comparison, a maximum 7-fold increase could be reached by stimulation of the native b-AR population. The receptor ratio between the rV2R and the native b-AR was '12:1 in both cardiomyoblasts and ventricular cardiomyocytes, although absolute expression levels of the receptor molecules were quite different in the 2 cell populations. Lower expression of rV2R in adult ventricular cardiomyocytes explains why, in these cells, AVP-dependent cAMP production did not reach the same level as in cardiomyoblasts. In both cell types, however, rV2R-dependent cAMP formation exceeded b-AR-dependent increases in cAMP. Noninfected cardiomyoblast membranes showed a basal 3 H-AVP binding, which was caused by the expression of native V1AR. Because no specific V1R antagonists exist, we differentiated both receptor subtypes in rV2R-expressing cells by addition of the selective V2R antagonist SR 121463A21 (Figure 2B). Taken together, Ad-V2R infection induced a genetic receptor subtype shift in both types of cardiomyocytes, overexpressing rV2R versus native V1R by a factor of 3:1.

The modulation of the endogenous cardiac signal transduction by the renal receptor led to an improvement of contraction amplitude in rat ventricular cardiomyocytes. Ad-V2Rinfected cardiomyocytes showed a dose-dependent increase in contraction amplitude to a maximum of 300% after stimulation with AVP. The half-maximal concentration for the AVP-stimulated cAMP response was '0.16 nmol/L, which corresponds to the peak AVP concentrations in vivo in the circulation of patients with congestive heart failure.7 Expression of the rV2R in the myocardium of these patients might allow to use the high systemic levels of AVP, which are involved in reducing ventricular contractility via V1R, for a beneficial, positive inotropic effect mediated by rV2R. The present study represents the first report about the functional modulation of the cardiac contractile apparatus by overexpression of a heterologous, positive inotropic receptor. It complements the attempts of earlier studies to improve cardiac contraction cycle by overexpressing calciumregulating proteins.24 In the failing myocardium, numerous alterations of the b-adrenergic signaling pathway occur. One approach that might be used to enhance cardiac function is to “resensitize” the b-adrenergic signaling cascade. Actually, this mechanism might explain at least part of the positive effect of some b-blockers, such as metoprolol, in the treatment of heart failure; these agents increased LV function and b-ARinduced contractility, and most probably, cardiac cAMP levels.25 A molecular tool to simulate this receptor resensitization might be the overexpression of a bARK-1 inhibitor protein (“bARKmini”), consisting of the carboxyl terminus

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Adenoviral Gene Transfer of the Human V2R Our approach was to investigate whether an ectopic expression of the V2R could be a sufficient alternative method to bypass the deficient b-adrenergic signaling pathway in cardiomyocytes. Because AVP levels in patients with heart failure vary with hemodynamic changes,7 cAMP would only be intermittently stimulated via rV2R when hemodynamic conditions are especially bad. rV2Rs were subject to endogenous mechanisms which promote homologous desensitization after short-term agonist exposure because we consistently observed an '20% loss of receptor function in both types of cardiomyocytes after short-term exposure to AVP. In contrast, the adenovirally introduced V2R was not submitted to receptor downregulation because we detected stable protein levels and no further reduction of cAMP responsiveness after prolonged agonist exposure in neither cardiomyoblasts nor adult cardiomyocytes. The reasons for this lack of downregulation cannot be determined, but it seems probable that the cytomegalovirus promotor used to drive receptor expression is not subject to endogenous mechanisms leading to receptor downregulation in cardiomyocytes. It remains to be seen whether this phenomenon also holds true for recombinant expression of other proteins.

Limitations of the Study Figure 6. Measurement of contraction amplitudes in ventricular cardiomyocytes. (A) Ad-V2R-infected (moi: 100 pfu/cell) or (B) Ad-bGal-infected (moi: 100 pfu/cell) cardiomyocytes were electrically paced as described in Methods. The cells were stimulated with increasing concentrations of AVP or isoproterenol (from 10 pmol/L to 0.1 mmol/L). Contraction amplitudes are shown as percent increase in reference to the baseline contraction amplitude before pharmacological intervention. The insets in A and B show the effect of SR 121463A (0.1 mmol/L) and propranolol (3 mmol/L) on the AVP- and isoproterenol-induced contraction amplitude in ventricular cardiomyocytes, respectively. AVP and isoproterenol were used in concentrations of 10 nmol/L and 0.1 mmol/L, respectively. Fractional shortening is shown as fold increase over basal values. The data represent mean6SEM of 10 independent experiments.

of bARK-1.26,27 Transgenic mice with overexpressed bARKmini in their hearts showed increased cardiac contractility and increased cAMP levels for the duration of their lives, without any signs of myocardial damage.26,28 bARKmini-transgenic mice also showed stable LV function after aortic banding, in contrast to the marked loss of LV contractility in their aortic-banded wild-type littermates.29 In contrast, other transgenic mice with specific molecular interventions in cardiac G protein-coupled signal transduction showed quite distinct characteristics: (1) overexpression of b2-AR led to mild cardiac fibrosis28 and (2) overexpression of Gas led to severe cardiac fibrosis and even cardiomyopathy.28 Taken together, specific molecular interventions in the G protein-coupled receptor system, leading to increased cAMP levels, might produce beneficial effects in the long run in the treatment of patients with heart failure, despite the failure of cAMPraising pharmacological agents, such as phosphodiesterase III inhibitors.30 Despite the disappointing results with these cAMP-raising drugs, a long-term beneficial effect of an intermittent stimulation of cardiomyocytic cAMP levels by a specific molecular mechanism might still be possible and should be further investigated.

It remains to be determined whether a similar biological effect can be observed in failing myocardium. Any approach to the treatment of heart failure will have to be verified by investigating a hemodynamic and survival benefit in animal models of congestive heart failure. Moreover, the capacity of enhanced cAMP levels to improve systolic dysfunction, despite alterations downstream of membranous cAMP formation, needs to be evaluated in the failing heart.

Acknowledgments We are thankful to Dr V. Sandig, MDC-Berlin, for donating the Ad-bGal and to Dr P. Goldsmith, NIH-Bethesda, for supplying an affinity-purified polyclonal antibody raised against the C-terminus of the human V2R. Additionally, we thank Sonja Fendt for excellent technical assistance in preparation of rat ventricular cardiomyocytes. This work was supported by the Deutsche Forschungsgemeinschaft, klinische Fo¨rderungsmittel der Technischen Universita¨t Mu¨nchen and Fonds der Chemischen Industrie to Gu¨nter Schultz.

References 1. Hepler JR, Gilman AG. G proteins. Trends Biochem Sci. 1992;17: 383–387. 2. Simon MI, Strathmann MP, Gautam N. Diversity of G proteins in signal transduction. Science. 1991;252:802– 808. 3. Birnbaumer M, Seibold A, Gilbert S, Ishido M, Barberis C, Antaramian A, Brabet P, Rosenthal W. Molecular cloning of the receptor for human antidiuretic hormone. Nature. 1992;357:333–335. 4. Thibonnier M, Preston JA, Dulin N, Wilkins PL, Berti-Mattera LN, Mattera R. The human V3 pituitary vasopressin receptor: ligand binding profile and density-dependent signaling pathways. Endocrinology. 1997; 138:4109 – 4122. 5. Garg R, Packer M, Pitt B, Yusuf S. Heart failure in the 1990s: evolution of a major public health problem in cardiovascular medicine. J Am Coll Cardiol. 1993;22(suppl A):3A–5A. 6. Cohn JN, Levine B, Olivari MT, Garberg V, Lura D, Francis GS, Simon AB, Rector T. Plasma noradrenaline as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med. 1984;311:819 – 823. 7. Landry DW, Levin HR, Gallant EM, Ashton RC, Seo S, D’Alessandro D, Oz MC, Oliver JA. Vasopressin deficiency contributes to the vasodilatation of septic shock. Circulation. 1997;95:1122–1125.

Downloaded from http://circ.ahajournals.org/ by guest on February 20, 2013

Laugwitz et al 8. Bristow MR, Ginsburg R, Minobe W, Cubicciotti RS, Sageman WS, Lurie K, Billingham ME, Harrison DC, Stinson EB. Decreased catecholamine sensitivity and b-adrenergic receptor density in failing human hearts. N Engl J Med. 1982;307:205–211. 9. Ungerer M, Bo¨hm M, Elce JS, Erdmann E, Lohse MJ. Altered expression of b-adrenergic receptor kinase and b1-adrenergic receptors in failing human heart. Circulation. 1993;87:454 – 463. 10. Kass-Eisler A, Falck-Pedersen E, Alvira M, Rivera J, Buttrick PM, Wittenberg BA, Cipriani L, Leinwand LA. Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and vivo. Proc Natl Acad Sci U S A. 1993;90:11498 –11502. 11. Johns DC, Nuss HB, Chiamvimonvat N, Ramza BM, Marban E, Lawrence JH. Adenovirus-mediated expression of a voltage-gated potassium channel in vitro (rat cardiac myocytes) and in vivo (rat liver). J Clin Invest. 1995;95:1152–1158. 12. Scho¨neberg T, Sandig V, Wess J, Gudermann T, Schultz G. Reconstitution of mutant V2 vasopressin receptors by adenovirus-mediated gene transfer: molecular basis and clinical implication. J Clin Invest. 1997; 100:1547–1556. 13. Bett AJ, Haddara W, Prevec L, Graham FL. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A. 1994;91:8802– 8806. 14. McGrory WJ, Bautista DS, Graham FL. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988;163:614 – 617. 15. Lieber A, Sandig V, Strauss MA. A mutant T7 phage promoter is specifically transcribed by T7-RNA polymerase in mammalian cells. Eur J Biochem. 1993;217:387–394. 16. Reithmann C, Scheininger C, Bulgan T, Werdan K. Exposure to the n-3 polyunsaturated fatty acid docosahexaenoic acid impairs a1-adrenoceptormediated contractile responses and inositol phosphate formation in rat cardiomyocytes. Naunyn-Schmiedebergs Arch Pharmacol. 1996;354: 109 –119. 17. Berger HJ, Prasad SK, Davidoff AJ, Pimental D, Ellingsen O, Marsh JD, Kelly RA. Continual electrical field stimulation preserves contractile function of adult ventricular myocytes in primary culture. Am J Physiol. 1994;266:H341–H349. 18. Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974;58:541–548.

February 23, 1999

933

19. Laugwitz K-L, Allgeier A, Offermanns S, Spicher K, Van Sande J, Dumont JE, Schultz G. The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families. Proc Natl Acad Sci U S A. 1996;93:116 –120. 20. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem. 1976;72:248 –254. 21. Serradeil-Le Gal C, Lacour C, Valette G, Garcia G, Foulon L, Galindo G, Bankir L, Pouzet B, Guillon G, Barberis C, Chicot D, Jard S, Vilain P, Garcia C, Marty E, Raufaste D, Brossard G, Nisato D, Maffrand JP, Le Fur G. Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist. J Clin Invest. 1996;12: 2729 –2738. 22. Delbridge LM, Connell PJ, Morgan TO, Harris PJ. Contractile function of cardiomyocytes from the spontaneously hypertensive rat. J Mol Cell Cardiol. 1996;28:723–733. 23. Drazner MH, Peppel KC, Dyer S, Grant AO, Koch WJ, Lefkowitz RJ. Potentiation of b-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes. J Clin Invest. 1997;2: 288 –296. 24. Hajjar RJ, Kang JX, Gwathmey JK, Rosenzweig A. Physiological effects of adenoviral gene transfer of sarcoplasmic reticulum calcium ATPase in isolated rat myocytes. Circulation. 1997;95:423– 429. 25. Gilbert EM, Abraham WT, Olsen S, Hattler B, White M, Mealy P, Larrabee P, Bristow MR. Comparative hemodynamic, LV functional, and antiadrenergic effects of chronic treatment with metoprolol vs. carvedilol in the failing heart. Circulation. 1996;94:2817–2825. 26. Koch WJ, Rockman HA, Samama P, Hamilton R, Bond RA, Milano CA, Lefkowitz RJ. Cardiac function in mice overexpressing the b-adrenergic receptor kinase or a bARK inhibitor. Science. 1995;268:1350 –1353. 27. Laugwitz K-L, Kronsbein K, Schmitt M, Hoffmann K, Seyfarth M, Scho¨mig A, Ungerer M. Characterization and inhibition of b-adrenergic receptor kinase in intact myocytes. Cardiovasc Res. 1997;35:324 –333. 28. Peppel K, Koch WJ, Lefkowitz RJ. Gene transfer strategies for augmenting cardiac function. Trends Cardiovasc Med. 1997;7:145–150. 29. Choi DW, Koch WJ, Hunter JJ, Rockman HA. Mechanism of b-adrenergic receptor desensitization in cardiac hypertrophy is increased b-adrenergic receptor kinase. J Biol Chem. 1997;272:17223–17229. 30. Packer M, Carver JR, Rodeheffer RJ. Effects of oral milrinone on mortality in severe chronic heart failure. N Engl J Med. 1991;325:1468 –1475.

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