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Am J Physiol Heart Circ Physiol 293: H2262-H2270, 2007. First published July 13, 2007; doi:10.1152/ajpheart.01320.2006
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Functional arginine vasopressin system in early heart maturation

Jolanta Gutkowska,1 Malgorzata Miszkurka,1 Bogdan Danalache,1 Natig Gassanov,1,2 Donghao Wang,1 and Marek Jankowski1

1Centre de Recherche, Centre Hospitalier de l'Université de Montréal, Hôtel-Dieu, Montreal, Quebec, Canada; and 2Department of Internal Medicine III, University of Cologne, Cologne, Germany

Submitted 4 December 2006 ; accepted in final form 2 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Since the neurohypophyseal hormone 8-arginine vasopressin (AVP) is involved in cardiovascular tissue hypertrophy and myocyte differentiation, it is possible that local AVP plays a role in heart maturation. AVP-specific RIA, RT-PCR, and immunoblot measurement of AVP receptors (VR) were used to investigate heart tissues from newborn and adult rats. To test AVP's role in differentiation and specialization into ventricle-like cardiomyocytes, we studied GFP-P19Cl6 stem cells, which express green fluorescence protein (GFP) reporter under transcriptional control of the myosin light chain-2v promoter. VR1 transcripts and proteins were higher in adult than in newborn rat hearts. In contrast, VR2 increased from postnatal day 1 to 5 and was barely detected in the adult rat heart. In cardiomyocytes expressing troponin C, immunofluorescence revealed VR2 and VR1. Intracellular cAMP increased 6.5- and 8.9-fold in response to the selective VR2 agonist 1-desamino-8-D-AVP (DDAVP) after 1 and 24 h, respectively. Cardiac AVP was high in 1- and 5-day-old (330 ± 26 and 276 ± 53 pg/mg protein, respectively) but low in 66-day-old (98 ± 15 pg/mg protein) rats. AVP immunostaining was detected in the tunica adventitia and endothelium of the coronary vessels. The possible role of AVP in cardiomyogenesis was indicated by DDAVP-AVP-dependent differentiation of GFP-P19Cl6 stem cells into contracting cells displaying GATA-4, a cardiac-specific marker, and ventricle-specific myosin light chain. Together, it is suggested that the AVP system is implicated in postnatal cardiac maturation.

vasopressin; heart maturation; vasopressin receptors; immunochemistry; cardiomyocyte differentiation

THE HYDROMINERAL BALANCE of the body and cardiovascular homeostasis are critically dependent on the action of 8-arginine vasopressin (AVP), a nonapeptide synthesized mainly in neurosecretory cells of the paraventricular and supraoptic nuclei of the hypothalamus and released via the posterior pituitary on stimulation by hyperosmolarity, hypotension, or hypovolemia (3). Its best-known action is the promotion of water conservation in the organism by increasing osmotic water permeability of the collecting ducts, allowing water to be reabsorbed. The effect on sodium is not evident, but most data suggest that AVP in vivo is antinatriuretic or might retard sodium excretion (4, 5).

AVP also belongs to the family of vasoactive and mitogenic peptides involved in physiological and pathological cell growth and differentiation. Its physiological activities are mediated through three receptor subtypes coupled to different G proteins: VR1 (V1a), VR2, and VR3 (V1b) (3, 17). VR1, which is abundant in vascular smooth muscles, causes vasoconstriction by increasing intracellular calcium via the phosphatidylinositol bisphosphonate cascade and a positive inotropic effect in cardiac muscle. Prolonged VR1 stimulation leads to the synthesis of proteins involved in cellular hypertrophy in vascular and myocardial tissues (28, 46). VR2, which is considered a renal receptor located on renal tubular epithelial cells, mediates water retention, as well as cAMP formation, and alters aquaporin (AQP) expression. VR3, which is localized in pituitary, pancreatic beta cells, and the adrenal medulla, induces the release of hormones.

The association of cardiac dysfunction with elevated plasma AVP levels has important prognostic implications (31). Indeed, experimental left ventricular hypertrophy in rats as a result of aortic stenosis increases AVP in magnocellular nuclei of the hypothalamus and heightens plasma AVP levels (27). Interestingly, AVP is secreted from isolated, pressure-overloaded buffer-perfused rat hearts; it has been localized in endothelial and vascular smooth muscle cells of arterioles and perivascular tissue (20). Therefore, AVP activity in the myocardium and coronary vasculature may be influenced by an intrinsic cardiac AVP system.

Oxytocin (OT), a peptide related to AVP, induces P19 stem cells (9, 21, 30, 40, 42) and stem cells present in the adult mouse heart (25) to differentiate into cardiomyocytes. However, the absence of OT or its receptor (OTR) in knockout mice has not been reported to produce cardiac insufficiencies (37). These findings, along with the fact that AVP promotes myogenic differentiation (35, 38), led us to hypothesize that AVP contributes to cardiac muscle maturation. Indeed, a recent study showed that AVP modifies spontaneous cardiomyocyte formation in embryoid bodies (EB) of D3 stem cells (11).

Here, we describe a functional AVP system in the newborn rat heart. Using GFP-P19Cl6 stem cells as a model (26), we also report the characteristics of cardiac AVP during early maturation and demonstrate its role in the induction of cardiomyogenesis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Experimental animals. The experiments were performed in accordance with guidelines of the Canadian Council on Animal Care with the approval of the Animal Care Committee of the Centre de Recherche, Centre Hospitalier de l'Université de Montréal. Pregnant (21 days) and nonpregnant female and male Sprague-Dawley rats were obtained from Charles River Laboratories (St.-Constant, QC, Canada). After delivery, 1-, 5-, 12-, and 21-day-old newborn and 66-day-old adult rats were killed by decapitation, and their hearts were excised rapidly and kept at –80°C for further processing.

Primary neonatal cardiomyocyte cell culture. Hearts were isolated from 2- to 3-day-old neonatal rats. Tissues, which contained both ventricles, were minced and trypsinized (50 µg/ml) overnight at 4°C. Subsequently, cardiomyocytes were dispersed by incubation in a collagenase type II buffer solution (Worthington Biochemical, Lakewood, NJ) at a concentration of 0.5 mg/ml for 45 min at 37°C, and the cellular suspension was passed through a polypropylene macroporous filter (105-µm mesh opening; Spectra Mesh, Spectrum Medical Industries, Houston, TX). The suspension was then centrifuged at 200 g for 5 min, and the cellular pellet was suspended in M199 with 10% FBS and penicillin-streptomycin (50 µg/ml; GIBCO-BRL, Burlington, ON, Canada). The cell suspension was preplated for 1 h at 37°C in 5% CO2 to decrease contamination of nonmuscle cells. Cell density was adjusted to 1 x 106 cells/ml using M199 supplemented with 10% FBS and penicillin-streptomycin. The cells were seeded in polystyrene, nonpyrogenic six-well culture plates (Becton-Dickinson, Franklin Lakes, NJ) precoated with 1% gelatin. They were then incubated in 5% CO2 at 37°C for 3–4 days. The purity of the primary neonatal cardiomyocytes was >95%, as assessed by beating phenotype of the cells.

Culture and differentiation of GFP-P19Cl6 cells. Experiments were performed in clonal derivatives of murine P19 embryonal carcinoma cell clone 6, which expresses a green fluorescence protein (GFP) reporter under transcriptional control of the myosin light chain-2v (MLC-2v) promoter. These GFP-P19Cl6 cells (a generous gift from Dr. C. Mummery, Hubrecht Laboratory, University Medical Centre, Utrecht, The Netherlands) were cultured as described elsewhere (26). Cells were grown in tissue culture dishes in complete {alpha}-modified Eagle's minimal essential medium ({alpha}-MEM, GIBCO-BRL) consisting of {alpha}-MEM supplemented with 2.5% heat-inactivated FBS, 7.5% heat-inactivated donor bovine serum (Cansera International, Rexdale, ON, Canada), and the antibiotics (GIBCO-BRL) penicillin (50 U/ml) and streptomycin (50 µg/ml). The cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and passaged every 2 days. For differentiation, drops containing 800 cells in a volume of 20 µl of complete medium were plated onto the lid of bacteriological-grade petri dishes (10 cm diameter) and incubated at 37°C for 2 days in the absence (noninduced) or presence of inducers: 10–6 M OT and 10–6 M AVP or 10–6 M 1-desamino-8-D-AVP (DDAVP). In some experiments, selective antagonists of VR1 [beta-mercapto-beta,beta-cyclopentamethylene-propionyl1,O-me-Tyr2,Arg8-vasopressin (H-5350)], VR2 [adamantaneacetyl1,O-Et-D-Tyr2,Val4,aminobutyryl6,Arg8,9-vasopressin (H-7705)], or OTR [beta-mercapto-beta,beta-cyclopentamethylene-propionyl-Tyr(Me)2-Ile-Thr-Asn-Cys-Pro-Orn-Tyr-NH2 (H-9405)] were added at 10–7 M to the AVP-containing differentiation media. The base of each culture dish contained PBS to prevent evaporation of the drops. All reagents were obtained from Bachem (Basel, Switzerland). After 2 days of incubation, EBs formed in the drops were transferred to bacterial dishes with fresh differentiation medium and kept for 3 more days in suspension in the absence (noninduced) or presence of inducers and antagonists. On day 5, EBs were collected and plated in complete {alpha}-MEM in tissue culture dishes in the absence of inducers until day 14. The medium was refreshed every 48 h. Cellular morphology and MLC-2v-GFP fluorescence were examined under an inverted microscope (Carl Zeiss, Jena, Germany) equipped for epifluorescence analysis. Micrographs were recorded with a Nikon Coolpix 5000 camera, and fluorescent areas were analyzed with Image J software (http://rsb.info.nih.gov/ij/, National Institutes of Health, Bethesda, MD), employing the threshold function.

RIA. Cardiac tissues were placed in acid solution (1 N HCl, 1% formic acid, 1% trifluoroacetic acid, and 1% NaCl) and homogenized, and 1 ml of homogenate was mixed with AVP extracted from heart tissue with heat-activated Vycor glass beads (catalog no. 7930 Mesh 140, Corning Glassworks, Corning, NY). The RIA was developed in our laboratory on the basis of previously published methodology (13) using specific antibodies generated against AVP (a generous gift from Dr. A. W. Cowley, Medical College of Wisconsin, Madison, WI). Synthetic AVP (Peninsula Laboratory, Belmont, CA) was labeled with 125I-Na by the lactoperoxidase method (13). Two hundred microliters of sample or standards (0–200 pg) were incubated with 100 µl of antibody (1:250,000dilution) for 24 h at 4°C. Then 100 µl of 125I-AVP (3,500 cpm) were added to each tube and incubated for 48 h at 4°C. Antibody-bound radioactivity was separated from free radioactivity after 2 h of incubation in the presence of 100 µl of normal rabbit serum, 100 µl of goat anti-rabbit globulin (1:50 dilution for each), and polyethylglycol (Sigma-Aldrich, Oakville, ON, Canada), with centrifugation at low speed for 30 min. Radioactivity in the supernatant was then measured in a gamma counter. Standard curves generated with 125I-AVP demonstrated a sensitivity of 0.1 pg and linearity in the range of 0.25–200 pg/ml. Cross-reactivity of the antibody was <1% with OT. The RIA was validated for accuracy and reproducibility. Serial dilutions (1:10, 1:20, and 1:40) of rat cardiac tissue homogenates produced competition curves, which were parallel to the standard (synthetic AVP), indicating that peptide content in the cardiac tissues has immunoreactivity similar to that of the synthetic peptide.

Western blot analysis. For Western blot analysis, frozen tissue samples were homogenized in Tris·HCl buffer containing a cocktail of protease inhibitors and insoluble materials removed by centrifugation at 4°C. After SDS-PAGE, the separated proteins were transferred to nitrocellulose membranes (Hybond-C, Amersham-Pharmacia, Baie d'Urfé, QC, Canada). Molecular size calibration was achieved with Broad Standard Solution (Bio-Rad Laboratories, Mississauga, ON, Canada). The nitrocellulose blots were blocked overnight with 5% nonfat milk in Tris-buffered saline (20 mM Tris·HCl, pH 8.0, 140 mM NaCl, 1% BSA, and 0.1% Tween 20) and then probed with rabbit anti-VR1, anti-VR3, or anti-VR2 antibody (Cedarlane Laboratories, Hornby, ON, Canada; 1:1,000 dilutions) for 2 h at room temperature. Antibody incubations and washes were performed in Tris-buffered saline. Detection was realized by enhanced chemiluminescence with an Amersham-Pharmacia ECL kit and an appropriate peroxidase-conjugated secondary antibody according to the manufacturer's protocol. Densitometry of the bands was quantitated by Image J software. Differences in protein loading were corrected by densitometric quantification of Ponceau-stained membranes.

Microscopy analyses. For immunocytochemistry (ICC), tissues were fixed by heart perfusion with 4% formaldehyde, embedded in wax, and cut into 5-µm sections. Rabbit antibodies against AVP were diluted 1:7,000. Staining was revealed by the biotin-streptavidin method (catalog no. 95-999, Histostain-DS, Zymed Laboratories, San Francisco, CA). Control staining, obtained by overnight preincubation of the anti-AVP antibody at 4°C in the presence of 10–6 M synthetic AVP or omission of primary antibodies, was negative, emphasizing ICC specificity.

For immunofluorescence, primary neonatal cardiomyocytes were grown on gelatin-coated microscopic glass slides for 5 days. The cells were then fixed in Zamboni solution for 45 min, washed with PBS, and kept in PBS-Triton 0.2% solution at 4°C before immunofluorescence staining. They were exposed in blocking solution (catalog no. 00-8020, Zymed Laboratories) and then incubated with respective rabbit antibody against rat VR1, VR2, or VR3 (1:500 dilutions; Cedarlane Laboratories). Then the cells were probed with Texas red-labeled goat anti-rabbit IgG conjugate (1:200 dilution; Ab 6793, Abcam, Cambridge, MA, http://www.abcam.com). Monoclonal antibody against cardiac troponin (1:100 dilution; Ab 7217-7, Abcam) was deployed for the identification of cardiomyocytes. To obtain green fluorescence, the secondary biotinylated rabbit antibody against mouse IgG (catalog no. BA-2001, Horse Vector Laboratories, Burlingame, CA; http://www.vectorlabs.com) was followed by streptavidin-Alexa Fluor 488 conjugate (catalog no. S11223 [GenBank] , Molecular Probes, Eugene, OR). To stain cell nuclei (blue), the 4',6-diamidino-2-phenylindole reagent was used in mounting solution (Prolong Gold reagent, catalog no. P36931 [UniProtKB/Swiss-Prot] , Molecular Probes). Controls stained without the primary antibody demonstrated no immunostaining of any cellular elements. Immunofluorescence was recorded under an inverted microscope (Eclipse TE 2000-S, Nikon) equipped with a Q Imaging QICAM-IR Fast 1394 digital charge coupled device camera. The Image J program was deployed to calculate green, red, and blue fluorescence areas, expressed as a percentage of the total image. Confocal microscopy images were produced in a microscope (model MRC1024, Bio-Rad, Microscience, Cambridge, MA) equipped with a krypton argon laser (excitation at 488 nm and 568 nm) combined with an inverted microscope (Eclipse TE 3000, Nikon) with emission filters measuring green and red fluorescence at wavelengths of 488 and 568 nm. Images were registered by Laser Sharp (version 3.2) software.

cAMP measurements. cAMP levels were measured in the cell media and extracts after incubation with AVP or DDAVP in the presence or absence of the phosphodiesterase inhibitor 2-isobutyl-1-methylxanthine (IBMX, 100 µM). Excess culture medium was aspirated. Cell monolayers were extracted in ice-cold 70% ethanol, and proteins were separated from the extracts by centrifugation. The extracts were dried in a Speedvac (Savant Instruments, Holbrook, NY) and resuspended in 0.05 M acetate buffer. cAMP was quantified in a Biotrak enzyme immunoassay system (model RPN225, Amersham-Pharmacia) according to the manufacturer's instructions.

RT-PCR. Total cellular RNA from cardiac tissue was extracted with Trizol reagent (Invitrogen Life Technologies, Burlington, ON, Canada) and converted to single-strand cDNA with use of avian myeloblastosis virus reverse transcriptase (Invitrogen Life Technologies), 4 µg of RNA, 4 µl of hexanucleotide primers (Amersham-Pharmacia), and a first-strand buffer in a final volume of 40 µl. To remove genomic DNA, RNA samples were incubated with deoxyribonuclease I (2 U/µg RNA; Invitrogen Life Technologies) for 30 min at 37°C. cDNA was amplified with the specific primers listed in Table 1. The samples were amplified by PCR (Robocycler Gradient 40 Thermocycler, Stratagene, La Jolla, CA) and resolved on agarose gel. Bands stained by ethidium bromide were analyzed with the Storm 840 imaging system and ImageQuant software (version 4.2, Molecular Dynamics, Sunnyvale, CA). To validate this RT-PCR assay as a tool for the semiquantitative measurement of mRNA, dose-response curves were established for different amounts of total RNA, and the samples were quantified in the linear phase of PCR amplification. These data were normalized to the corresponding values of 18S RNA PCR products serving as internal controls (Ambion, Austin, TX). Specificity of the PCR products was analyzed in a sequencing genetic analyzer (model 3100, Applied Biosystems, Foster City, CA) according to the manufacturer's procedure described in the sequencing kit (Big Dye Terminator, version 3.1).


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  		Table 1. PCR primer sequence

 
Statistical analysis. Values are means ± SE of a minimum of five different animals. Statistical comparisons were made using Prism 3 (GraphPad Software, San Diego, CA). Data from the RIA studies were analyzed by one-way ANOVA. Differences were assessed by the Newman-Keuls multiple comparisons test and examined with the linear trend formula. Comparisons between two groups were conducted by unpaired Student's t-test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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AVP system identification during rat heart maturation. RIA revealed the presence of AVP in the rat heart. The radioactivity values plotted from serial dilutions (1:10, 1:20, and 1:40) of rat cardiac tissue homogenates from atria and ventricles produced competition curves parallel to the standard (synthetic AVP), indicating similar immunoreactivity (Fig. 1A). AVP levels were high in newborn rat hearts (330 ± 26 and 276 ± 52 pg/mg protein on postnatal days 1 and 5, respectively) and low in adult rat hearts on postnatal day 66 (98 ± 15 pg/mg protein, P < 0.01, n = 10; Fig. 1B). RIA analysis of dissected heart chambers from female and male rats on postnatal day 21 (Fig. 1C) did not disclose sex-related differences in AVP concentration. The results demonstrated that cumulative (males and females) AVP concentration was 229 ± 24 and 222 ± 26 pg/mg protein in the right and left atrium, respectively. These values were significantly higher than those in the left and right ventricle (37 ± 3 and 79 ± 6 pg/mg protein, respectively, n = 13, P < 0.001).


Figure 1
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  		Fig. 1. Detection and characterization of arginine vasopressin (AVP) in rat heart during postnatal maturation. A: AVP immunoreactivity in serial dilutions (1:80, 1:40, and 1:20) of rat extracts from right atrium (RA) and right ventricle (RV) homogenates measured by RIA. Results were compared with AVP standard curve. Ordinate shows ratio (expressed as percentage) of bound 125I-labeled AVP in the presence (B) to that in the absence (B0) of synthetic vasopressin. B: changes in AVP concentration in rat heart on postnatal days 1, 5, and 66 measured by RIA (n = 6–12). *P < 0.05. C: AVP concentration in heart chambers of female and male rats on postnatal day 21 (n = 6–12). LA, left atrium; LV, left ventricle. *P < 0.05.

 
Control staining of aorta sections from adult rats indicated that the tunica adventitia is the major site of AVP immunoreactivity (Fig. 2A). AVP has also been found in the endothelial cell layer of the tunica intima and in microvessels of the tunica media (Fig. 2A); the AVP-preadsorbed antibodies did not stain these aortic structures (Fig. 2, B). In the heart, strong staining with anti-AVP antibody was found in the tunica adventitia and less staining in the tunica intima of the coronary vessels (Fig. 2C). No AVP-specific staining was demonstrated in cardiac myocytes (Fig. 2D).


Figure 2
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  		Fig. 2. AVP immunocytochemical staining in rat heart and aorta. A: AVP in adventitia (Adv) and endothelium (En) of adult female rat aorta. Med, tunica media. B: significant reduction of AVP immunostaining in the aorta after preincubation of anti-AVP antibodies with synthetic AVP. C: AVP staining in the tunica adventitia of coronary arteries and endothelium (arrows) of cardiac section from rat on postnatal day 12. AVP immunoreactivity was absent in the myocardium (D). Scale bars, 50 µm.

 
AVP receptors in newborn and adult rats. The observation that cardiac AVP is elevated during early heart maturation led us to characterize cardiac AVP receptors to understand possible autocrine and paracrine AVP actions. RT-PCR mRNA analysis, indicating local receptor transcription in the heart, demonstrated the presence of VR1 and VR2 in the heart of newborn animals on postnatal days 1 and 5, as well as in adults on postnatal day 66 (Fig. 3, A and B). We were unable to detect VR3 mRNA in the postnatal rat heart. AVP VR1 mRNA in the rat heart increased between postnatal days 1 and 5 and persisted at a high level in adult rats (Fig. 3A). In contrast, the cardiac VR2 transcript (Fig. 3B) was higher on postnatal day 5 than on postnatal day 1 (2-fold) and postnatal day 66 (1.6-fold). Western blotting revealed VR1 and VR2 proteins at the expected molecular weights of 45 and 62 kDa, respectively (Fig. 3, C and D). VR1 protein (Fig. 3C) was higher in adult animals on postnatal day 66 than in newborn rats. VR2 protein increased in the heart from postnatal day 1 to day 5 but declined in the mature rat heart (Fig. 3D).


Figure 3
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  		Fig. 3. Detection and characterization of AVP receptors (VR1 and VR2) in rat heart during early postnatal maturation. A and B: RT-PCR analysis of VR1 and VR2 mRNA expression in rat heart on postnatal days 1 and 5 and in adult rats on day 66. Left lane: molecular weight ladder (123 bp). Levels of specific mRNA bands were normalized to intensity of the corresponding 18S mRNA bands. C and D: Western blot analysis of 50-kDa VR1 and 62-kDa VR2 proteins in rat heart. Ponceau S-stained membranes were used to normalize protein loading on gels. *P < 0.05 vs. preceding age group.

 
VR1 (Fig. 4A) and VR2 (Fig. 4C) were expressed in culture of cardiomyocytes isolated from rats on postnatal day 3. Immunofluorescence demonstrated positive staining for VR1 and VR2 in cells expressing the cardiomyocyte marker troponin C. Staining for VR3 was negative (Fig. 4B). VR2 visibility in cardiomyocytes was enhanced by immunofluorescent staining combined with confocal microscopy (Fig. 4D). Intensity analysis suggested higher expression of VR2 than VR1 (16.2 ± 1.6 vs. 6.7 ± 0.6% of culture areas) in images of similar fractions displaying troponin C and 4',6-diamidino-2-phenylindole coloration (Fig. 4E).


Figure 4
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  		Fig. 4. Representative immunofluorescence images of vasopressin receptors (stained red by Texas red conjugate) in isolated rat newborn cardiomyocytes expressing troponin C (TrC) marker (sstained green by Alexa Fluor 488 conjugate). Cell nuclei were visualized by 4,6-diamino-2-phenylindole (DAPI). A, B, and C: staining with anti-VR1, anti-VR3, and anti-VR2 antibodies, respectively. D: confocal microscopy image of VR2. Arrows indicate receptor staining. E: comparative quantification of areas emitting each color in cells stained for vasopressin receptors. Values are means ± SE (n = 6). Scale bars, 50 µm. *Significant difference between image areas occupied by VR1 and VR2 (P < 0.001). There were no differences in corresponding areas occupied by troponin C and cell nuclei.

 
To identify the signaling potential of VR2 in the newborn heart, we determined whether AVP and the VR2-selective analog DDAVP increase cAMP in newborn rat cardiomyocytes in culture. As demonstrated in Fig. 5, AVP and DDAVP strongly stimulated intracellular cAMP and its release into cardiomyocyte culture medium. In cells incubated with 10–6 M DDAVP + 100 µM IBMX, a significant cAMP increase was also observed in cardiomyocyte extracts: DDAVP induced cAMP elevations of 6.5- and 9-fold after 1 and 24 h, respectively (Fig. 5A). In the medium (Fig. 5B), cAMP rose 48-fold after 1 h and 30-fold after 24 h compared with cells incubated with IBMX alone (n = 6, P < 0.001).


Figure 5
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  		Fig. 5. AVP and 1-desamino-8-D-AVP (DDAVP, a specific AVP agonist of VR2) induce cAMP release in neonatal rat cardiomyocyte culture stimulated for 1 or 24 h in the presence of 100 µM IBMX (n = 6). A: intracellular cAMP. B: cAMP released into medium. c, Control. *P < 0.05 vs. control. &P < 0.05, AVP vs. DDAVP.

 
AVP stimulation of beating cell colonies in stem cells. To identify the possible cardiomyogenic function of the AVP system in the heart, we tested its effect on the differentiation of GFP-P19Cl6 cells. The expression of AVP receptors was investigated in noninduced GFP-P19Cl6 cells propagated in nondifferentiating conditions (day 0) in GFP-P19Cl6 EB kept for 4 days in suspension (day 4) and then in GFP-P19Cl6 cells growing after EB plating on days 6, 8, 10, and 12. As illustrated in Fig. 6, GFP-P19Cl6 cells growing in EB expressed all three transcripts for AVP receptors. In nondifferentiated GFP-P19Cl6 cells growing in monolayers, VR1 and VR2, but not VR3, transcripts were detected. The VR3 transcript was induced in the EB stage on day 4 (Fig. 6C). Then, after cell plating, the VR1 transcript was barely detectable (Fig. 6A), VR2 mRNA was found at a similar level at all time points, and VR3 increased until day 8 and was maintained at this level on days 10 and 12 (Fig. 6B).


Figure 6
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  		Fig. 6. RT-PCR analysis of VR1 (A), VR2 (B), VR3 (C), and 18S (D) genes in GFP-P19Cl6 cells. Receptor transcripts were investigated in noninduced GFP-P19Cl6 cells propagated in nondifferentiating conditions (day 0), in GFP-P19Cl6 embryoid bodies (EBs) kept for 4 days in suspension (day 4), and then in GFP-P19Cl6 cells growing after EB plating on days 6, 8, 10, and 12. H, RNA control sample from mouse heart; MW, molecular weight ladder (123 bp).

 
Exposure of GFP-P19Cl6 cells to AVP resulted in formation of contracting cells (Fig. 7A) and induction of the cardiomyocyte-specific transcription factor GATA-4 (Fig. 7B). Fluorescence microscopy was employed to visualize ventricle-specific MLC-2v-GFP staining intensity and distribution in response to differentiation inducers (Fig. 7C). Basal fluorescence was seen only in noninduced cultures (Fig. 7C1). Cultures treated with OT (Fig. 7C2), AVP (Fig. 7C3), and DDAVP (Fig. 7C4) produced intense fluorescence areas of MLC-2v induction. AVP induction of MLC-2v-GFP-expressing cells was reduced in the presence of OTR, VR1, and VR2 antagonists, as shown by quantitative analysis in Fig. 7C and illustrated in Fig. 7C5 using, as an example, VR2 inhibition.


Figure 7
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  		Fig. 7. AVP induces cardiomyocyte differentiation in mouse embryonal carcinoma cell line GFP-P19Cl6. A: oxytocin (OT)- and AVP-mediated induction of contracting colonies of GFP-P19Cl6 cells. B: AVP induces GATA-4 mRNA in GFP-P19Cl6 cells at a level comparable to that induced by OT. Blot shows GATA-4 mRNA levels reflected by 275-bp bands adjusted to intensity of corresponding 18S mRNA bands. C: green fluorescent protein (GFP) quantitative image analysis of GFP-P19Cl6 cells induced by OT, DDAVP, and AVP in the presence or absence of OT or AVP antagonists for VR1 (VP1) and VR2 (VP2). NI, noninduced. Values are means ± SE of 3 independent studies. *P < 0.05. C1–C5: representative GFP reporter expression under transcriptional control of the MLC-2v promoter in noninduced P19 cells, OT-, AVP-, and DDAVP-induced P19 cells, and AVP-induced P19 cells in the presence of the VR2 antagonist.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study follows up reports on the mechanism of effects on the heart mediated by OT (9, 21, 25, 30) and AVP (11, 20, 46). We have provided the first evidence that the local cardiac AVP system is activated during early rat maturation. RIA revealed elevated AVP concentrations in the newborn heart, localized predominantly in the coronary vessels. The AVP targets in newborn rats were VR1 and VR2 expressed in cardiomyocytes. Another novel finding in the present study is the identification and full characterization of VR2 in newborn hearts, confirmed by RT-PCR at the transcript level, with specific VR2 protein shown by immunoblotting and localized by immunofluorescence in newborn cardiomyocytes, as well as by cAMP production in these cells in response to DDAVP treatment. Finally, using the experimental model of cardiomyogenesis, GFP-P19Cl6 stem cells, we demonstrated that AVP induces the cardiomyocyte differentiation pathway, as disclosed by the formation of beating cell colonies and cardiac gene expression.

In contrast to the rat hypothalamus (1, 36, 41), where AVP expression increases with age, cardiac AVP levels decline significantly from the postnatal period to adult life, suggesting different roles in physiology. AVP is present in the circulation of homozygous Brattelboro (di/di), centrally AVP-deficient, and hypophysectomized rats (3). These observations, along with the fact that AVP in the circulation is below the level required to elicit physiological actions, raised the hypothesis of local AVP production in peripheral organs. Indeed, Hartman et al. (14) noted an increment of circulating AVP in newborn rats, despite low AVP concentration in the hypothalamus and pituitary gland. Later, AVP synthesis was detected by Hupf et al. (20) in pressure-overloaded, isolated, perfused rat hearts. These authors reported that AVP released into the coronary effluent may increase the circulating AVP pool. The heart may contribute to elevated AVP plasma levels in cardiac failure (12, 24, 31, 33) and possibly in conditions of left ventricular dysfunction after myocardial infarction (34). Consistent with these findings, we found that AVP is elevated in newborn rat hearts, which indicates the heart's contribution to increased AVP levels in the newborn circulation (14).

Our ICC studies revealed localization of cardiac AVP in rats mainly in the tunica adventitia and endothelium of the coronary vessels and the presence of AVP receptors in cardiomyocytes. A VR1 population involved in Ca2+ mobilization and myocyte hypertrophy has been previously shown in neonatal rat cardiomyocytes (10, 46). These data suggest a possible role for AVP in the postnatal physiology of cardiac muscles.

It has been originally established that VR2, which promotes the activation of adenylyl cyclase, is physiologically expressed in only the kidneys and is absent in the myocardium of adult animals. However, VR2 is not restricted to cells of the collecting ducts, as was believed, and some data indicate extrarenal VR2 expression. Hirasawa et al. (16) described VR2 in various regions of the brain, with predominant expression in the hippocampus. In the present study, we have demonstrated cardiac VR2 by RT-PCR and Western blotting in the heart and cardiomyocytes of newborn rats, despite an earlier report that the receptor's mRNA is absent in the heart of this species. Our data are in agreement with those of Kaufmann et al. (22), who noted VR2 expression in the human heart. In addition, these authors demonstrated activation of nitric oxide (NO) production in human endothelial cells by DDAVP, suggesting vasodilatation through VR2. Indeed, AVP has also been shown to cause vasodilatation in numerous vascular beds (6, 43), distinguishing this hormone from other vasoconstrictor agents. An alternative mechanism of AVP-induced vasodilatation has been proposed: the activation of endothelial OTR triggers the activation of endothelial isoforms of NO synthase (39, 44).

In the present study, cAMP production was demonstrated in cardiomyocytes exposed to AVP and DDAVP for 1 or 24 h. These results indicate VR2 signaling in these cells, even with prolonged agonist exposure. This long-lasting VR2 activity in the presence of agonist has been described in rat cardiomyocytes subjected to adenoviral transfer of the VR2 gene (23). It has been reported, in addition, that VR2 overexpression in the rat myocardium increases cardiac contractility in vivo (45), consistent with the notion that cAMP is the most important modulator of cardiac contractility (47). Therefore, we hypothesize that cardiac-specific enhancement of VR2 signaling may play a role during significant changes in the contractility and histological structure of the ventricular myocardium during the early neonatal period (2). Indeed, in cardiomyocytes and many other cell types, cAMP mediates the action of a number of different receptors and modulates many cellular functions as diverse as movement, growth, metabolism, and synaptic plasticity (19).

AVP can target AQPs, channel proteins responsible for the water permeability of plasma membranes via VR2. VR2-activated AQPs can contribute to the dramatic increase of cell volume during development of heart hypertrophy. Therefore, cardiac VR2 activation early in postnatal life may play a role in regulating cardiomyocyte volume. AQP-1 exists in cardiomyocyte caveolae (29); therefore, it is likely that cAMP activates AQP-1 and augments cell volume. Two observations support this hypothesis. 1) Patil et al. (32) demonstrated AVP-cAMP-dependent water permeability in oocytes overexpressing AQP-1. 2) Chen et al. (8) found that AVP enhances water permeability in astrocytes, producing cellular edema.

The observation that the rat cardiac AVP system is strongly activated around birth evoked the hypothesis that AVP is involved in cell differentiation early in the neonatal period (2). Age-related changes in expression of AVP receptors were also established by RT-PCR in noninduced GFP-P19Cl6 EBs. The results demonstrated that all three receptors were abundantly expressed in the early stage (days 4–6) of EBs. However, we cannot exclude the possibility that loss of VR1 transcripts and the appearance of VR3 transcripts may represent mRNA contributions/alterations from noncardiac cells in EBs. Our data show that exogenous provision of AVP into EBs is sufficient to trigger cardiomyogenesis, as demonstrated by its ability to differentiate stem cells into cardiomyocytes displaying such characteristics as GATA-4 expression, MLC-2v-GFP-associated fluorescence, and spontaneous beating. Since VR2 expression remained high at all differentiation periods, we used DDAVP, a specific VR2 agonist, to induce cardiomyogenesis in GFP-P19Cl6 cells. The results disclosed that, similar to AVP, DDAVP treatment of GFP-P19Cl6 cell EBs induced beating cell colonies and MLC-2v-GFP expression. The possibility that VR2 is involved in cardiomyocyte differentiation is consistent with recent findings of dose-dependent cAMP increases in spontaneous cardiomyocyte differentiation in EBs of murine D3 stem cells (7). As we reported recently, AVP modulates this process by augmenting cardiomyocyte yield and promoting selection of the ventricular electrophenotype (11). The AVP-mediated increase in endothelial NO synthase expression also seems to be partly regulated by OTR, inasmuch as lower endothelial NO synthase mRNA and protein levels were observed in D3 cells treated with OTR antagonist (11). In addition, some physiological AVP effects are thought to be mediated by OTR, inasmuch as AVP receptors and OTR share high primary sequence homology (17), and cross talk between the two systems has been shown. However, our experiments suggest low OTR involvement in the AVP-mediated cardiac effects due, probably, to the lower affinity of AVP for OTR and, especially, DDAVP compared with VR1 and VR2. It is noteworthy that there is considerable diversity of OT-mediated signaling pathways, including cAMP activation and cGMP-mediated growth stimulation with calcium increases (48), which corroborates AVP signaling mediated by VR1 and VR2 (17). The specific pathway that activates cardiomyogenesis is unknown. However, enhanced GATA-4 expression, consistently observed throughout cardiac differentiation in response to AVP in the present and previous studies (11) and after OT exposure of stem cells for inducing differentiation in P19 cells (9, 30, 40) and somatic cardiac stem cells (25) or for increasing cardiomyocyte yield in EBs (15), might contribute significantly to the cardiomyogenic actions of both peptides.

In summary, the physiological relevance of AVP's effect on early heart maturation is indicated by several findings. 1) High levels of immunoreactive AVP, present in the newborn heart, decline in adulthood. 2) AVP is present in coronary vasculature, and this observation, together with the finding of AVP in the mammalian sympathetic nervous system (18), may cast light on whether AVP enhances cardiac muscle maturation. 3) In addition to functional OTR (9, 15, 21, 25, 30), stem cells express AVP receptors and differentiate to cells expressing markers of cardiomyocytes in response to AVP treatment. Thus it is interesting to speculate that, during embryonic development, AVP may interact with the OT system in the physiological control of cardiomyogenesis. In conclusion, our data support the view that the cardiac AVP system plays a role in the early stages of heart maturation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported by Canadian Institutes of Health Research Grant MOP-53217 (to J. Gutkowska and M. Jankowski) and Canadian Heart and Stroke Foundation Grant NET SRD-63193 (to J. Gutkowska and M. Jankowski).


   ACKNOWLEDGMENTS
 
We acknowledge the editorial work of Ovid Da Silva (Research Support Office, Research Centre, Centre Hospitalier de l'Université de Montréal).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Gutkowska, Centre de Recherche CHUM, Hôtel-Dieu, 3850 St-Urbain, Montréal, QC, Canada H2W 1T7 (e-mail: jolanta.gutkowska{at}umontreal.ca)

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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