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First published online October 18, 2007
Stem Cells Vol. 26 No. 1 January 2008, pp. 45 -54
doi:10.1634/stemcells.2007-0289; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

Functional Activity of the Carboxyl-Terminally Extended Oxytocin Precursor Peptide During Cardiac Differentiation of Embryonic Stem Cells

Natig Gassanova,b, Dominic Devostc, Bogdan Danalacheb, Nicolas Noiseuxb, Marek Jankowskib, Hans H. Zinggc, Jolanta Gutkowskab

aDepartment of Internal Medicine III, University of Cologne, Cologne, Germany;
bResearch Centre, Centre Hospitalier de l'Université de Montréal Hôtel-Dieu, Montreal, Quebec, Canada;
cDepartment of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

Key Words. Embryonic stem cells • Developmental biology • Gene transfer • Growth factors

Correspondence: Correspondence: Jolanta Gutkowska, Ph.D., CHUM-Hôtel-Dieu Research Center, 3850 Saint Urbain, Montreal, Quebec H2W 1T7, Canada. Telephone: 514-890-8000, ext. 12731; Fax: 514-412-7204; e-mail: jolanta.gutkowska{at}umontreal.ca

Received on April 20, 2007; accepted for publication on October 2, 2007.

First published online in STEM CELLS EXPRESS  October 18, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The hypothalamic post-translational processing of oxytocin (OT)-neurophysin precursor involves the formation of C-terminally extended OT forms (OT-X) that serve as intermediate prohormones. Despite abundant expression of the entire functional OT system in the developing heart, the biosynthesis and implication of OT prohormones in cardiomyogenesis remain unknown. In the present work, we investigated the involvement of OT-X in cardiac differentiation of embryonic stem (ES) cells. Functional studies revealed the OT receptor-mediated cardiomyogenic action of OT-Gly-Lys-Arg (OT-GKR). To obtain further insight into the mechanisms of OT-GKR-induced cardiac effects, we generated ES cell lines overexpressing the OT-GKR gene and enhanced green fluorescent protein (EGFP). The functionality of the OT-GKR/EGFP construct was assessed by fluorescence microscopy and flow cytometry, with further confirmation by radioimmunoassay and immunostaining. Increased spontaneously beating activity of OT-GKR/EGFP-expressing embryoid bodies and elevated expression of GATA-4 and myosin light chain 2v cardiac genes indicated an inductive effect of endogenous OT-GKR on ES cell-derived cardiomyogenesis. Furthermore, patch-clamp experiments demonstrated induction of ventricular phenotypes in OT-GKR/EGFP-transfected and in OT-GKR-treated cardiomyocytes. Increased connexin 43 protein in OT-GKR/EGFP-expressing cells further substantiated the evidence that OT-GKR modifies cardiac differentiation toward the ventricular sublineage. In conclusion, this report provides new evidence of the biological activity of OT-X, notably OT-GKR, during cardiomyogenic differentiation.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Oxytocin (OT), a neurohypophyseal nonapeptide, has long been known for its role as a female reproductive hormone by stimulating milk ejection and prolactin release and inducing uterine contractions. In previous studies, we have provided evidence for a new role of OT as a cardiomyogenic factor and demonstrated the existence of an entire functional OT/OT receptor (OTR) system in the heart [1, 2]. However, knowledge of OT ontogeny in the developing heart is still limited. A large number of studies on the development of the hypothalamo-neurohypophyseal system have revealed that OT is mainly produced as a large precursor molecule in two hypothalamic nuclei, the supraoptic nucleus and paraventricular nucleus [3]. The initial translation product from the OT gene was identified as OT-Gly-Lys-Arg-neurophysin I [4]. During post-translational processing, neurophysin I is cleaved, and proteolytic cleavage of OT-Gly-Lys-Arg (OT-GKR) tripeptide by carboxypeptidase results in the release of OT-Gly-Lys (OT-GK) and OT-Gly (OT-G). Finally, OT-G is converted by {alpha}-amidating enzyme to mature amidated OT. The peptides OT-GKR, OT-GK, and OT-G are referred to as carboxyl-extended forms of OT (OT-X) [5].

Recent studies have suggested the potential physiological role of these intermediate forms. Thus, the existence of OT-X has been shown in the uterus [6]. Increased blood levels of OT-X, but not of biologically active, amidated OT, have been found in response to estrogen treatment and associated with blood pressure reduction [7, 8]. Interestingly, OT-X concentrations in fetal sheep plasma are 35-fold higher than hormonally active OT in the early stages of development [5]. Similarly, the amidated OT form is not detectable in the fetal rat brain until embryonic day 21, despite abundant expression of the principal intermediate form, OT-GKR, during the same time period [9].

The objective of the present work was to explore the involvement of OT-X in cardiac development. Here, we describe the profound cardiomyogenic actions of both exogenously applied OT-GKR and OT-GKR precursor peptide, delivered by OT-GKR gene transfer in embryonic stem (ES) cells. This report provides new evidence of the biological activity of OT-X, notably OT-GKR, during cardiomyogenic differentiation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Generation of DNA Constructs
A signal peptide OT-GKR-coding cassette was generated by the annealing of sense and antisense oligonucleotides, each covering a portion of the coding region extended at its 3' end by a stretch of overlapping complementary sequences. Both oligonucleotides bore a BglII restriction site at their 5' ends for cloning purposes and, also, an upstream Kozak sequence and a downstream stop codon for proper translation. The sequences of the oligonucleotides used were as follows: 5'-gaagatctgccgccaccatggcctgccccagtctcgcttgctgcctgc-ttggcctactggctctgacctccgcctgctacatc-3' and 5'-gaagatcttcacctcttgccgcccagggggcagttctggatgtagcaggcggaggtcag-3'. Single-stranded DNA overhangs were filled in using the Klenow fragment, and the resulting double-stranded DNA was digested by BglII and ligated into the murine stem cell virus-internal bicistronic expression-enhanced green fluorescent protein vector (pMSCV-IRES-EGFP) [10] at the single BglII site located upstream of the IRES-EGFP cassette. The construct was verified for proper directional insertion and sequence integrity by polymerase chain reaction (PCR) and DNA sequencing. To insert the OT-GKR-IRES-EGFP cassette into the vector pcDNA3.1(+)/ZEO for the establishment of stable cell lines, the DNA fragment between the AflII located upstream of the OT-GKR cassette and the unique BamHI site located downstream of the EGFP coding region was excised from the recombinant vector pMSCV-OT-GKR-IRES-EGFP and ligated to the pcDNA vector between the unique AflII and BamHI restriction sites of its multiple cloning site (Fig. 1A).


Figure 1
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  		Figure 1. Schematic structure of pcDNA3.1/ZEO-OT-GKR-IRES/EGFP (A) and pcDNA3.1/ZEO-IRES/EGFP (B) constructs. Abbreviations: CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; OT, oxytocin.

 
To generate the appropriate control pcDNA vector lacking the OT-GKR coding region, the corresponding AflII/BamHI fragment from the original nonrecombinant pMSCV-IRES-EGFP vector was cloned into the pcDNA vector as described above (Fig. 1B). The absence of the OT-GKR cassette was confirmed by DNA sequencing.

Both constructs were stably transfected into ES cells with a FuGENE 6 kit (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) according to the manufacturer's instructions. The transfected ES cells were cultured for 10 days in the presence of Zeocin, and resistant clones were further selected. ES cells transfected with pcDNA3.1/ZEO-IRES/EGFP served as controls in the experiments with transgenic cell lines.

Cell Culture and Differentiation
To generate ventricular-specific ES cells, the cytomegalovirus (CMV)enhanced/myosin light chain 2V (MLC-2V)/green fluorescent protein (GFP) construct (a generous gift from Dr. C.L. Mummery, Hubrecht Laboratory, University Medical Centre, Utrecht, The Netherlands) was stably transfected into D3 ES cells with a FuGENE 6 kit (Roche) according to the manufacturer's protocol. The transfected ES cells were cultured for 10 days in the presence of neomycin (G418), and resistant clones were further selected. ES cells of the D3 cell line and its transgenic OT-GKR/EGFP, control EGFP, and MLC-2V/GFP-expressing derivatives were cultivated in Dulbecco's modified Eagle's medium supplemented with 1,000 units/ml leukemia inhibitory factor (LIF; Chemicon, Temecula, CA, http://www.chemicon.com), 15% fetal calf serum (FCS), penicillin/streptomycin, nonessential amino acids, GlutaMAX (Invitrogen, Burlington, ON, Canada, http://www.invitrogen.com), and β-mercaptoethanol (Invitrogen). For the induction of differentiation, ES cells were cultured without LIF in hanging drops to form embryoid bodies (EBs) for 2 days, then kept in suspension for an additional 3 days, and finally plated on gelatin-coated 24-well plates for n days (designated dn). To examine the effect of OT-X on cardiac differentiation, EBs were exposed to 10–6 M OT (Bachem, Torrance, CA, http://www.bachem.com) or 10–6 M OT-Gly, OT-Gly-Lys, OT-Gly-Lys-Arg (synthesized by the Peptide Synthesis and Protein Sequencing Core Facility of Eastern Quebec, Quebec City, QC, Canada) in the presence or absence of a selective OTR antagonist (OTA; 10–6 M β-mercapto-β,β-cyclopentamethylene-propionyl-Tyr(Me)2-Ile-Thr-Asn-Cys-Pro-Orn-Tyr-NH2) at the time of differentiation induction in hanging drops. In additional experiments, all test peptides were applied at the time of EB plating to determine whether treatment time affects cardiac differentiation. The media were changed daily, and OT and its precursors were added from a frozen stock for a single experiment. A micromolar concentration was applied, since preliminary dose-response studies with OT and OT-X revealed that the 10–6 M concentration was the most efficient in inducing the beating activity of EBs.

Intracellular Ca2+ Measurements
Intracellular calcium concentration ([Ca2+]i) measurements were performed by Fura-2 ratiometric analysis [11]. Single cells were obtained by dissection of spontaneously beating EB areas at day (d) 7–d8 after plating and enzymatic dispersion with Accutase (Innovative Cell Technologies Inc., San Diego, CA, http://www.innovativecelltech.com). The cell suspension was pipetted onto glass coverglass and assayed 1 day later. For Fura-2 loading, the cells were incubated in phosphate-buffered saline (PBS) containing the acetoxymethyl ester form of Fura-2 (Fura-2 AM; Invitrogen) for 30 minutes at 37°C and then washed twice. The coverglass was placed into a flow chamber on the stage of an inverted fluorescence microscope. Fura-2-loaded cells were exposed to alternate illumination at 340 and 380 nm with a high-pressure mercury lamp via interference filters (Chroma Technology, Rockingham, VT, http://www.chroma.com) mounted on a filter wheel (Sutter Instrument, Novato, CA, http://www.sutter.com) and a dichroic mirror (510/540 nm; Chroma Technology). Fluorescence images were recorded during a 100-millisecond exposure at 15-second intervals with a digital camera. The data were analyzed with MetaFluor software (Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com).

Radioimmunoassay
For radioimmunoassay (RIA), 50 control or OT-GKR-expressing (OT-GKR/EGFP+) EBs were plated on gelatin-coated dishes. Differentiation media, containing 20% FCS and other supplements, were collected 48 hours after the last medium change and directly analyzed for OT-GKR concentration by RIA [1]. To exclude the possible interference of peptide contained in the differentiation media, OT-GKR protein levels were determined in the culture media without EBs (negative controls). Polyclonal rabbit antibody specifically recognizing OT-GKR peptide [12] (VA18; a generous gift from Dr. H. Gainer, Laboratory of Neurochemistry, NIH, Bethesda, MD) was applied to measure OT-GKR concentration.

Flow Cytometry
Dissociation of OT-GKR/EGFP+ and MLC-2V/GFP+ EB outgrowths to single-cell suspensions was performed with Accutase on the days indicated. The isolated cells were washed and suspended in PBS and then filtered with a sterile nylon cell strainer (Falcon; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). A minimum of 10,000 viable cells were acquired for each sample and quantified by passage in a FACSAria cell sorter (BD Biosciences). Nontransfected ES cells served as negative controls. The sorted OT-GKR/EGFP+ cells were collected in corresponding differentiation media and allowed to reattach to culture dishes for at least the next 24 hours for functional studies. The BD Biosciences CellQuest software was used for data acquisition and data analysis.

Immunostaining and Fluorescence Microscopy
Green fluorescent cells were incubated in gelatin-covered Lab-Tek plates (Nunc, Rochester, NY, http://www.nuncbrand.com) for 48 hours after flow cytometry sorting. The cells were fixed in a solution containing 4% paraformaldehyde in 0.1 M PBS, pH 7.4, for 20 minutes. Subsequently, they were washed in 0.1 M PBS, permeabilized for 10 minutes with 0.4% Triton X-100, and labeled with a rabbit antibody raised against OT-GKR peptide [12]. After washing steps with 0.1% Triton X-100 and PBS, the samples were incubated with secondary Texas Red-conjugated anti-rabbit antibody (Invitrogen) and analyzed by confocal microscopy (MRC1024; Bio-Rad, Cambridge, MA, http://www.bio-rad.com). The ImageJ program (NIH) was applied to calculate green and red fluorescence.

Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from fluorescent cells with TRIzol reagent (Invitrogen). First-strand cDNA was synthesized in a final volume of 40 µl containing first-strand buffer, 3 µg of cellular RNA, 4 µl of hexanucleotide primers (GE Healthcare, Baie d'Urfe, QC, Canada), and avian myeloblastosis virus reverse transcriptase (12 units/µg of RNA; Invitrogen). For all PCR studies, dose-response curves were established for different amounts of RNA, and the samples were quantified in the linear range of PCR amplification. These values were normalized to corresponding 18S mRNA. To exclude contamination from genomic DNA, a control experiment was undertaken for each RNA sample without reverse transcriptase before PCR. Primer sequences and conditions for the reverse transcription (RT)-PCR analysis of 143-base pair (bp) GATA-4 (32 cycles), 286-bp MLC-2a (30 cycles), and 189-bp MLC-2v (30 cycles) mouse transcripts have already been described [13–15]. The PCR products were size-fractionated by 2% agarose gel electrophoresis and quantified with the Storm 840 imaging system and ImageQuant software (version 4.2; GE Healthcare, Sunnyvale, CA).

Immunoblotting
For Western blot analysis, enzymatically dispersed cells were lysed with radioimmunoprecipitation assay buffer. Protein concentration was quantified spectrophotometrically by Bradford assay (Bio-Rad). Separated proteins were transferred electrophoretically to polyvinylidene difluoride membranes (GE Healthcare). Blots were incubated overnight at 4°C with the primary antibody against mouse connexins (Cx) 43 and 45 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), followed by incubation with peroxidase-conjugated secondary antibody (Amersham-Pharmacia) the next day for 1 hour at room temperature. Cx proteins were visualized with an ECL kit (GE Healthcare).

Electrophysiology
Action potential (AP) recordings were obtained on isolated, beating cardiac myocytes in whole-cell patch-clamp configuration at room temperature with an Axopatch 200B amplifier (Axon Instruments/Molecular Devices Corp., Foster City, CA, http://www.moleculardevices.com) while sampling at 10 kHz and filtering at 2 kHz, as previously described in more detail [16]. The recording bath solution contained 135 mmol/l NaCl, 5 mmol/l KCl, 2 mmol/l CaCl2, 1 mmol/l MgCl2, 10 mmol/l glucose, and 10 mmol/l HEPES; pH was adjusted to 7.4 with NaOH. Borosilicate microelectrodes were pulled with tip resistances of 2–4 M{Omega} (Narishige Inc., Tokyo, http://www.narishige.co.jp/niusa/index.htm).

Statistical Analysis
The results are presented as means ± SEM. Comparisons between groups were evaluated by one-way analysis of variance. p < .05 values were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Implication of OT-X in Cardiomyogenic Differentiation
In the initial set of experiments, we assessed the effect of various OT-X isoforms on ES cell-derived cardiogenic differentiation. EBs were analyzed for their beating activity at d5 and d12 after EB plating (n = 120 in each group). Compared with control EBs, no significant changes were observed in EBs exposed to OT-G and OT-GK (Fig. 2). Application of amidated OT increased spontaneous contraction incidence, but this effect was significant only at d12 (37.6% ± 4.0% vs 23.4% ± 4.1%; p < .05) and not significant at d5 (40.2% ± 2.8% vs 35.2% ± 3.5%). In contrast, a strong increase in the number of beating EBs was noted in OT-GKR-treated ES cells at both d5 (49.2% ± 3.4%; p < .05) and d12 (40.2% ± 2.8%; p < .05).


Figure 2
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  		Figure 2. Effect of OT and C-terminally extended OT forms on spontaneously beating EB activity at day 5 (upper panel) and day 12 (lower panel). More beating EBs were observed in response to OT-GKR treatment at both investigated developmental stages, whereas a significant OT effect was detected only in the later differentiation stage. Application of a selective OTA effectively inhibited the cardiomyogenic actions of both peptides. *, p < .05 versus control. Abbreviations: OT, oxytocin; OTA, oxytocin receptor antagonist.

 
To ascertain the involvement of OTR in OT-GKR-mediated cardiomyogenic actions, OT- and OT-GKR-treated EBs were also exposed to 10–6 M OTA. Indeed, OTA application inhibited the effect of both OT (24.4% ± 4.0% at d12; p < .05) and OT-GKR (d5: 39.2% ± 4.5%; d12: 31.0% ± 3.4%).

We also evaluated whether application time of the test substances would affect the course of cardiac differentiation. For this purpose, EBs (n = 120 in each group) were treated with OT, OT-G, OT-GK, and OT-GKR at the time of EB plating, and the number of beating EBs was analyzed. Interestingly, administration of OT or its precursors at the time of EB plating did not significantly affect beating activity at d5 (OT, 36.7 ± 5.8; OT-G, 28.3 ± 4.8; OT-GK, 35.8 ± 4.6; OT-GKR, 41.7 ± 5.4; p = NS vs. control EBs) or d12 (OT, 25.8 ± 3.1; OT-G, 23.3 ± 3.6; OT-GK, 19.2 ± 3.8; OT-GKR, 33.3 ± 5.7; p = NS).

OT-GKR Induces [Ca2+]i Release
The inhibition of EB beating activity by OTA indicated OTR-mediated functional activity of OT-GKR in differentiating ES cells. Since [Ca2+]i mobilization represents a hallmark of OTR activation [17], we evaluated OT and OT-GKR effects on [Ca2+]i concentrations. Ratiometric Fura-2 fluorescence analysis of single cells revealed [Ca2+]i elevation in response to both OT and OT-GKR (Fig. 3). Additional treatment with OTA abolished OT- and OT-GKR-induced [Ca2+]i mobilization, confirming the involvement of OTR in OT-GKR-mediated signaling.


Figure 3
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  		Figure 3. Application of 10–6 M OT or OT-GKR peptides at t = 0 minutes induced increased intracellular Ca2+ release in cardiac myocytes. The peak of the Ca2+ response was effectively blocked in the presence of OTA. The results are representative of four independent experiments. *, p < .05 versus control. Abbreviations: OT, oxytocin; OTA, oxytocin receptor antagonist.

 
Functional Characterization of OT-GKR/EGFP-Expressing ES Cells
Guided by the above-described findings, the cardiogenic potency of OT-GKR was further studied in ES cell lines stably expressing the OT-GKR/EGFP construct. Generally, EGFP expression was observed in both beating and nonbeating areas of EBs in a ratio of 1:1 to 1:2, with no significant differences in fluorescence intensities within EBs. The EGFP expression pattern was analyzed quantitatively by flow cytometry on EBs at d2, d7, and d12. Typically, the highest percentage of EGFP+ cells was observed at d7, following a decrease with further differentiation (Fig. 4A–4C). In addition, we noted an enhanced number of spontaneously contracting OT-GKR/EGFP+ EBs compared with control EBs at d7 (49.4 ± 4.6 vs. 28.2 ± 4.8; p < .05; n = 72) (Fig. 4D).

The functionality of the OT-GKR/EGFP construct was confirmed by the determination of endogenous OT-GKR synthesis in transgenic EBs by RIA. Basal peptide concentration measured in culture media without EBs was 28.6 ± 8.5 pg/ml (n = 5). OT-GKR peptide levels in media secreted from control EBs at d5 and d12 were 194.5 ± 24.0 pg/ml and 130 ± 8.3 pg/ml, respectively (n = 6). Corresponding values obtained from OT-GKR/EGFP+ cells disclosed a significant increment of peptide concentration at both investigated developmental stages (d5: 513.3 ± 72.6 pg/ml, p < .05, n = 6; d12: 358.2 ± 61.6 pg/ml, p < .001, n = 6) (Fig. 5A). The specificity of the OT-GKR/EGFP construct was further proven by immunostaining with antibody recognizing OT-GKR peptide. As shown in Figure 5B–5G, significantly higher peptide expression was detected in labeled OT-GKR/EGFP+ cells compared with control cells. Colocalization studies revealed that 76% ± 4.8% of EGFP+ cells were positively stained for OT-GKR, whereas this percentage was lower in control samples (11% ± 1.4%).


Figure 4
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  		Figure 4. Characterization of OT-GKR/EGFP-expressing cells. (A): Typical fluorescence profile of OT-GKR/EGFP+ embryonic stem cells at d7, as detected by flow cytometry analysis. (B): Nontransfected cells were used to set background fluorescence. (C): Percentages of EGFP+ cells in various developmental stages at d2, d7, and d12. *, p < .05 versus d2. (D): Increased number of beating EBs expressing the OT-GKR/EGFP construct compared with control EBs. *, p < .05 versus control. Abbreviations: d, day; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; OT, oxytocin.

 


Figure 5
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  		Figure 5. OT-GKR release and expression in differentiating embryonic stem cells (ES). (A): Radioimmunoassay revealed increased OT-GKR peptide levels in media secreted from OT-GKR/EGFP+ EBs. (B–G): Confocal images of OT-GKR-stained OT-GKR/EGFP+ (B–D) and control (E–G) differentiating embryonic stem cells. Images of green fluorescent protein (left panels) and OT-GKR positively stained (red, middle panels) cells at d5. Right panels: Merged colocalization views confirmed that the majority of OT-GKR/EGFP+ displayed significant OT-GKR staining. Scale bar = 10 µm. Abbreviations: d, day; EGFP, enhanced green fluorescent protein; OT, oxytocin.

 
OT-GKR Enhances Expression of GATA-4 and MLC-2v Cardiac Genes
Functional studies on spontaneously beating activity suggested a cardiomyogenic potential of OT-GKR expressed endogenously in ES cells. To characterize the cardiac gene expression profiles of OT-GKR/EGFP+ ES cells, the mRNA transcript levels of GATA-4 and the cardiac chamber-specific marker genes MLC-2a and MLC-2v were analyzed at various stages of differentiation by RT-PCR. As illustrated in Figure 6A and 6B, consistent induction of GATA-4 expression was found throughout cardiac differentiation. Enhanced MLC-2v gene expression was also detected in differentiating OT-GKR/EGFP+ ES cells. In contrast, there were virtually no differences in MLC-2a mRNA levels between control and OT-GKR/EGFP+ cells, pointing to a lineage-specific cardiomyogenic action of OT-GKR peptide.


Figure 6
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  		Figure 6. Cardiac cell markers expressed in OT-GKR/EGFP-transfected embryonic stem cells (ES). (A, B): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of GATA-4, myosin light chain (MLC)-2v, and MLC-2a gene expression in OT-GKR/EGFP+ embryonic stem (ES) cells. Densitometric analysis of band intensities (B) revealed enhanced expression of GATA-4 and MLC-2v genes in OT-GKR/EGFP+ cells, whereas MLC-2a mRNA levels were unaffected by endogenous OT-GKR. 18S mRNA was used as internal CTR. Three separate experiments yielded similar results. (C): Consistent with the RT-PCR results, quantitative flow cytometry analysis revealed more MLC-2v/GFP+ terminally differentiated ES cells. *, p < .05 versus CTR. Abbreviations: CTR, control; d, day; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; M, molecular weight (123-base pair DNA ladder); OT, oxytocin.

 
Guided by the above-described findings, the impact of OT-GKR was further determined on specialized cardiac ventricular phenotypes using MLC-2v/GFP-expressing ES cells. The MLC-2v gene has been demonstrated in several studies to exhibit ventricular tissue-restricted expression in embryonic and P19 stem cells [18, 19]. MLC-2v/GFP-expressing ES cells were treated with 10–6 M OT-GKR peptide at the onset of differentiation, and flow cytometry was applied to quantify the effect of OT-GKR. In line with the RT-PCR results, a significant increase in the number of MLC-2v/GFP+ cells was observed in response to OT-GKR treatment in terminally differentiated cardiac cells (18% ± 1.9 vs 7.5% ± 1.3 in the nontreated group; p < .05) (Fig. 6C).

OT-GKR Directs the Development of ES Cells Toward a Ventricular Phenotype
To examine the electrophysiological properties of OT-GKR/EGFP-expressing cardiomyocytes and to evidence the cardiac subtype-specific effects of OT-GKR peptide, patch-clamp recordings were undertaken on spontaneously contracting single cells. Since the AP pattern provides a clear fingerprint of the cardiac cell subtype, we measured the AP configurations of nonfluorescent (EGFP–) (n = 28) and green fluorescent (EGFP+) (n = 31) cardiomyocytes. Based on their basic electrophysiological properties, pacemaker-, ventricular-, and atrial-like cells could be identified among differentiated EGFP– cardiac cells (Fig. 7A). Pacemaker-like cells (n = 7) were characterized by a vigorous spontaneous beating rate (168 ± 12.7 beats per minute), a short overshoot (14 ± 1.6 mV), and a less negative resting membrane potential (–52.7 ± 4.6 mV). AP duration, measured from maximal depolarization to 90% repolarization (APD90), was 110 ± 8.4 milliseconds. Atrial-like cells (n = 9) displayed typically shorter APD90 (82 ± 5.7 milliseconds) and higher overshoots (31 ± 4.4 mV). Similarly, higher amplitudes with more negative resting membrane potentials were also recorded in ventricular-like cells (n = 12). However, the pronounced plateau phase along with the longest APD90 (228 ± 14.1 milliseconds) was a prominent hallmark of ventricular-like cells, clearly distinguishing them from other cell phenotypes.


Figure 7
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  		Figure 7. Electrophysiological characterization of OT-GKR-expressing cardiomyocytes. (A): Typical pacemaker-like (1), atrial-like (2), and ventricular-like (3) action potential (AP) profiles in spontaneously contracting EGFP– cardiomyocytes. Similar atrial- and ventricular-like AP profiles were recorded in EGFP+ cardiomyocytes. (B): More cells exhibiting a ventricular AP profile were observed in OT-GKR/EGFP+ cardiomyocytes. No EGFP+ pacemaker-like phenotype was detected by patch-clamp. (C): Similar results were obtained for cardiomyocytes treated with 10–6 M OT-GKR peptide, indicating a potential role of OT-GKR in inducing cardiomyogenesis toward the ventricular cell lineage. (D): In line with the electrophysiological results, Cx43 protein was significantly elevated in OT-GKR-expressing embryonic stem cells. Cx45 protein levels tended to be downregulated in transgenic EBs. β-Actin served as internal control. *, p < .05 versus control. Abbreviations: Cx, connexin; EGFP, enhanced green fluorescent protein; OT, oxytocin.

 
AP recordings in EGFP+ cells revealed no significant differences in AP shape or the major electrophysiological properties of atrial- and ventricular-like cells compared with EGFP– cardiac cells. Interestingly, no pacemaker-like phenotype was detected by patch-clamp in EGFP+ cells. In addition, strong induction of cells with the ventricular AP profile (n = 25, or 80% of all investigated EGFP+ cardiac myocytes) was evident in OT-GKR/EGFP+ cardiomyocytes compared with EGFP– cells (43%), indicating the potential of OT-GKR to induce cardiomyogenesis toward the ventricular cell phenotype (Fig. 7B).

To exclude the possibility that transgene OT-GKR expression is present only in ventricular cardiomyocytes and that OT-GKR is silent in other cardiac subtypes, additional electrophysiological characterization was performed on single cells treated with OT-GKR peptide (n = 26). Nontreated cardiac myocytes were used as controls (n = 19). Similar to the results obtained in transgene OT-GKR+ cells, chronic exposure to OT-GKR increased the percentage of ventricular cells (n = 7, or 37%, in controls vs. n = 20, or 77%, in OT-GKR-treated cells; p < .05) without affecting their basic electrophysiological characteristics (Fig. 7C).

To further examine the lineage-specific effect of endogenous OT-GKR in cardiomyogenesis, we analyzed Cx43 and Cx45 protein levels in OT-GKR/EGFP+ EBs. Both connexins exhibited a specific expression pattern in the mouse heart, with Cx43 being a principal gap junction protein in the ventricular working myocardium [20], whereas Cx45 was abundantly expressed in specialized cardiac conduction tissue [21]. In line with the electrophysiological results, Cx43 protein was significantly increased in OT-GKR/EGFP+ ES cells, whereas Cx45 protein levels tended to be downregulated, as demonstrated in Figure 7C.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
This study shows, for the first time, the functional activity of the C-terminally extended OT precursor peptide OT-GKR during ES cell-derived cardiomyogenesis and suggests a new role for OT-GKR in cardiac development. Furthermore, stable transfection of the pcDNA3.1/ZEO-OT-GKR-IRES/EGFP construct in ES cells enabled the identification of OT-GKR-producing cell populations. Patch-clamp recordings revealed an increased subgroup of ventricular-like OT-GKR-expressing and OT-GKR-treated cardiac myocytes. Upregulated Cx43 protein expression in OT-GKR/EGFP+ cells further substantiated the evidence that OT-GKR modifies the differentiation of cardiac precursor cells toward the ventricular sublineage.

In earlier reports, we provided evidence that an entire OT system exists in the heart [1, 2, 22], and OT induces the differentiation of P19 stem cells [13, 15, 22]. In this study, we investigated the involvement of OT precursor peptides in cardiomyogenesis, using ES cells, a validated in vitro model of cardiac differentiation [14, 23]. Among all OT-X, OT-GKR was the only isoform to exhibit prominent cardiomyogenic actions in functional experiments in response to exogenous peptide application. The dominance of OT-GKR effects compared with those of other OT-X forms could be explained by OT-GKR not being proteolytically cleaved during early cardiomyogenesis, or its cleavage late in cardiac development. The significance of this finding happens remains unclear and might also involve different precursor storage of cleaved proteins in cardiac tissue. Interestingly, similar dominance of OT-GKR expression exists in the brain, and the increased ratio of OT-GKR/other OT-X and OT-GKR/OT persists into late postnatal stages [9].

Ca2+ mobilization studies showed functional OT-GKR and OT activity. Although similar [Ca2+]i induction was seen for OT in human vascular cells, no reports so far have elucidated the OT or OT-GKR effects on [Ca2+]i in cardiomyocytes. The observed sustained effect on [Ca2+]i may be due to the nature of both peptides. In support of this notion, prolonged and long-lasting Fura-2 mobilization of [Ca2+]i was demonstrated in cardiac cells in response to arginine vasopressin (AVP) [24, 25], another neuropeptide from the posterior pituitary, which has more than 80% homology with OT [26] and exhibits significant affinity for OTR [27]. On the other hand, single cells in our study were obtained by mechanical dissection of spontaneously beating EB areas following enzymatic dispersion, as described previously [14, 16]. Therefore, we cannot exclude the possibility of having a small percentage of other noncardiac cells in [Ca2+]i mobilization studies, which may contribute to the prolonged increase of [Ca2+]i in response to OT or OT-GKR peptides.

Functional tests involving OTA revealed that it did not reduce the number of OT-GKR-stimulated beating EBs to control levels. This indicates the presence of other signaling pathways in response to OT-GKR. Because of high receptor homology between OT and AVP receptors [27], it appears likely that (at least some) AVP receptors may be involved in OT-GKR-mediated pathways. Further support for the potential involvement of AVP receptors in OT-GKR-linked pathways is provided by the finding that AVP receptors V1a and V2 are highly expressed and functionally active during ES cell-derived cardiomyogenesis [14]. To elucidate the role of endogenous OT-GKR in cardiac differentiation pathways and to investigate the precise electrophysiological profile of OT-GKR-expressing cells, ES cell lines overexpressing the OT-GKR gene were generated. Zeocin-driven antibiotic resistance was used for clonal expansion, and the EGFP reporter gene enabled the selective identification of transgenic cells in vitro.

The expression of OT-GKR coding cDNA was driven by a CMV promoter. Reports about CMV promoter efficiency for the genetic manipulation in ES cells are controversial. In general, data indicate lower CMV promoter activity in ES cells compared with other cell types [28, 29]. One likely explanation is that ES cells may express only a limited number of genes while maintaining a multipotential developmental capacity [30, 31]. ES cells may not express some transcriptional (co)factors necessary for CMV promoter activity and, thus, cannot support its function. In addition, differential CMV behavior may be due to the developmental and cellular heterogeneity of the differentiating ES cell culture system [32].

The choice of vector, transfection system, procedure, and reporter gene could explain some differences in transfection efficiencies in ES cells using the CMV promoter [29, 33]. Indeed, the finding that IRES-dependent second gene expression is 20%–50% that of the first gene in a bicistronic vector [34] could also explain the decreased number of EGFP+ cells in our study. On the other hand, the relatively low number of EGFP+ cells is likely the result of enzymatic treatment of EBs for flow cytometry analysis.

Overall, OT-GKR effects are to some extent similar to OT-induced cardiac differentiation. Also, the involvement of OTR and the cardiac gene expression profile of both inducers strengthen this notion [13, 15]. However, it is unlikely that OT, which can emerge as a result of endogenous cleavage of OT-GKR, mediates this effect, since RIA identified OT-GKR peptide in media from OT-GKR-expressing EBs. Prominent anti-OT-GKR immunostaining confirmed specific OT-GKR expression in OT-GKR/EGFP+ cells. The specificity of antibodies applied for immunocytochemistry and RIA in the present work has been characterized extensively [12]. Despite the high specificity for OT-GKR, we do not exclude some cross-reactivity with OT or other OT precursors, which could explain the nonspecific staining. On the other hand, the fact that some nontransfected cells were also stained positively with OT-GKR antibody may indeed indicate endogenous OT-GKR expression in these cells.

The choice of the construct may explain why not all EGFP+ cells react with OT-GKR antibody. It is likely that not all cells possess the cell machinery necessary for OT-GKR synthesis while at the same time expressing CMV promoter-driven EGFP. In addition, lower OT-GKR peptide stability compared with EGFP and, thus, increased susceptibility to degradation during the preparation procedure for immunocytochemistry might be other reasons for the relatively low percentage of stained cells.

Functional studies on EB beating activity indicated an even more powerful cardiomyogenic potential of OT-GKR in comparison with the fully processed OT molecule, since OT-GKR application enhanced the number of contracting EBs in both the early and late steps of cardiomyogenesis, suggesting the full functionality of OT-GKR precursor peptide, whereas the post-translational machinery of OT processing is not completely mature in the earlier steps of cardiac differentiation [15]. On the other hand, the induction of cardiomyogenesis by both amidated OT and OT-GKR to a similar extent at a later developmental stage indicates that the C-terminal portion of the hormone may not be essential for terminal cardiomyocyte maturation.

Enhanced cardiac differentiation in OT-GKR/EGFP+ EBs was also reflected in the consistent upregulation of GATA-4 expression. Increased MLC-2v mRNA levels along with unchanged MLC-2a gene expression provided an initial sign of potential cellular diversification by endogenous OT-GKR, given the lineage-specific expression pattern of MLC-2v and MLC-2a cardiac genes [35, 36]. Indeed, patch-clamp analysis of AP recordings revealed the predominance of ventricular phenotypes among OT-GKR-expressing cardiomyocytes. Since no pacemaker-like ES cells were detected in late-stage cardiomyocytes, it appears likely that OT-GKR modifies the commitment of cardiac precursor cells toward ventricular cell subtypes. In support of this notion, OT-GKR/EGFP+ EBs displayed increased expression of Cx43 protein. Previous reports have demonstrated the specific spatiotemporal expression pattern of Cx isoforms in the working myocardium and cardiac conduction tissue. Cx43 is the only Cx isotype known to be expressed in the adult working myocardium [20, 37]. Consistently, Cx43 is virtually absent in the developing atria and central conduction system [38, 39]. In contrast, Cx45 expression is preferentially associated with specialized cardiac conduction tissue [21, 40, 41]. However, Cx45 expression in the atrial and ventricular myocardium has also been reported [41]. In addition, Cx45 has been detected in endocardial endothelial cells [42]. Hence, in view of the diversity of spatiotemporal Cx45 expression, the observed tendency toward decreased Cx45 protein in OT-GKR/EGFP+ EBs in the present work might be the result of differential Cx45 regulation and distribution by OT-GKR in various heart compartments due to divergent peptide effects on the differentiation of cardiac conduction tissue and the ventricular working myocardium.

The potential of OT-GKR to promote the ventricular phenotype is of direct interest in the development of cell therapies for the heart, especially hearts injured by infarction, since this condition adversely affects ventricular myocytes [43]. In addition to promoting the cardiomyogenic differentiation of ES cells, OT-GKR peptide might have the potential to induce the cardiac differentiation of somatic stem cells of the adult heart, as these cells have been shown to respond in such a way to OT itself [44, 45].

In summary, the present study demonstrates, for the first time, that the OT precursor molecule OT-GKR has profound cardiomyogenic action that can be of physiological importance in cardiac development. It also points to its potential use, in addition to OT, in the development of therapeutic strategies for the heart. In view of our findings on cardiogenesis, it is possible that OT-GKR, a stable and abundantly expressed OT-X isoform in mammalian hypothalamo-neurohypophyseal tissue [9], may also play a pivotal role in fetal brain development. However, further investigation is necessary to gain more insights into the biological and functional significance of OT-GKR and other OT-X in brain and cardiac development.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Dr. Sylvain Gimmig for assistance with FACS. We acknowledge the editorial work of Ovid Da Silva (Research Support Office, Research Centre, Centre Hospitalier de l'Université de Montréal) and the excellent secretarial help of Jennifer Lelievre. This study was supported by grants from the Deutsche Forschungsgemeinschaft and Koeln Fortune Program/Faculty of Medicine, University of Cologne (to N.G.) and the Canadian Institutes of Health Research (NET Program), as well as the Heart and Stroke Foundation (to M.J. and J.G.).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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