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Oxytocin-Gly-Lys-Arg: a novel cardiomyogenic peptide.

Danalache BA, Gutkowska J, Slusarz MJ, Berezowska I, Jankowski M - PLoS ONE (2010)

Bottom Line: In embryonic P19 cells, OT-GKR induced contracting cell colonies and ventricular CM markers more potently than OT, an effect being suppressed by OT antagonists and OTR-specific small interfering (si) RNA.The V1a receptor antagonist and specific si-RNA also significantly reduced OT-GKR-stimulated P19 contracting cells.These results raise the possibility that C-terminally extended OT molecules stimulate CM differentiation and contribute to heart growth during fetal life.

View Article: PubMed Central - PubMed

Affiliation: Research Centre, Centre Hospitalier de l'Université de Montréal - Hôtel-Dieu, Montreal, Quebec, Canada.

ABSTRACT

Background: Oxytocin (OT), synthesized in the heart, has the ability to heal injured hearts and to promote cardiomyogenesis from stem cells. Recently, we reported that the OT-GKR molecule, a processing intermediate of OT, potently increased the spontaneous formation of cardiomyocytes (CM) in embryonic stem D3 cells and augmented glucose uptake in newborn rat CM above the level stimulated by OT. In the present experiments, we investigated whether OT-GKR exists in fetal and newborn rodent hearts, interacts with the OT receptors (OTR) and primes the generation of contracting cells expressing CM markers in P19 cells, a model for the study of early heart differentiation.

Methodology/principal findings: High performance liquid chromatography of newborn rat heart extracts indicated that OT-GKR was a dominant form of OT. Immunocytochemistry of mouse embryos (embryonic day 15) showed cardiac OT-GKR accumulation and OTR expression. Computerized molecular modeling revealed OT-GKR docking to active OTR sites and to V1a receptor of vasopressin. In embryonic P19 cells, OT-GKR induced contracting cell colonies and ventricular CM markers more potently than OT, an effect being suppressed by OT antagonists and OTR-specific small interfering (si) RNA. The V1a receptor antagonist and specific si-RNA also significantly reduced OT-GKR-stimulated P19 contracting cells. In comparison to OT, OT-GKR induced in P19 cells less α-actinin, myogenin and MyoD mRNA, skeletal muscle markers.

Conclusions/significance: These results raise the possibility that C-terminally extended OT molecules stimulate CM differentiation and contribute to heart growth during fetal life.

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Molecular docking of 3-D models of activated human OTR and V1aR with OT/OT-GKR peptides obtained by the MolDock Optimizer algorithm from Molegro Virtual Docker.(A) The front upright view position (side view) of the OTR-OT- GKR complex structure. (A1) The section (rectangle) shown in panel A1 from the top a intracellular view (i.e. rotation by 90° out of plane) of the marked section in A, demonstrate OT-GKR (green) in active conformation inside the OT binding site (the transmembrane helices in red and the cavity in violet). (B) side view of the V1aR-OT- GKR complex, (B1) V1aR top view. (C1) detail of docking view of OTR-OT-GKR complex, and (C2) detail of V1aR-OT-GKR complex. D displays the schematic model of human OTRs with marked amino acid residues that are putatively involved in ligand-binding. The amino acid residues in black circles have been proposed as OT docking sites, and the red bars represent docking sites of OT-GKR. (E) Schematic model of human vasopressin V1aR binding with OT-GKR and OT. Amino acid residues are identified by a 1-letter code in Table 1.
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pone-0013643-g002: Molecular docking of 3-D models of activated human OTR and V1aR with OT/OT-GKR peptides obtained by the MolDock Optimizer algorithm from Molegro Virtual Docker.(A) The front upright view position (side view) of the OTR-OT- GKR complex structure. (A1) The section (rectangle) shown in panel A1 from the top a intracellular view (i.e. rotation by 90° out of plane) of the marked section in A, demonstrate OT-GKR (green) in active conformation inside the OT binding site (the transmembrane helices in red and the cavity in violet). (B) side view of the V1aR-OT- GKR complex, (B1) V1aR top view. (C1) detail of docking view of OTR-OT-GKR complex, and (C2) detail of V1aR-OT-GKR complex. D displays the schematic model of human OTRs with marked amino acid residues that are putatively involved in ligand-binding. The amino acid residues in black circles have been proposed as OT docking sites, and the red bars represent docking sites of OT-GKR. (E) Schematic model of human vasopressin V1aR binding with OT-GKR and OT. Amino acid residues are identified by a 1-letter code in Table 1.

Mentions: Because of significant molecular differences between OT-GKR and OT, the question has been raised whether OT-GKR interacts with OTR binding sites. For this reason, we performed computational docking analysis of OT molecules in OTR binding sites. Figure 2 illustrates the docking of 3-D human OTR with OT-GKR and OT-modeled molecules in front upright view (Fig. 2A) and a view from the extracellular side (Fig. 2B). Three conformations for both OT-GKR and OT were analyzed. Six related receptor-Gα-segment-OT-GKR complexes were obtained. Possible hydrophobic and electrostatic interaction points in dynamic complexes of these molecules were indicated by estimated binding affinity energies of −6.6±0.4 kJ/mol for OT-GKR and −11.8±0.6 kJ/mol for OT. Using distance criteria, the program identified receptor amino acid residues interacting with ligands. The essential hydrogen bond and strong electrostatic interactions between both OT molecules and the receptors were characterized by visual inspection. The results are reported in Figure 2D and Table 1. Several amino acid residues have been proposed to interact with OT and OT-GKR in the OTR model where the red bars represent docking with OT-GKR, and the black bars indicate docking with OT molecule (Fig. 2D). Docking to OTR in positions V115, K116, Q119, M123, Q171, F185, T205, Y209 and Q295 was noted for both OT and OT-GKR models (Table 1). These docking positions constituted 42% of all observed docking sites of OT-GKR. Among OT-GKR interactions, the special notice should be given to binding of the arginine-12 (R12), (Fig. 2 D) to OTR. We also analyzed the docking of OT-GKR and OT molecules in the 3-D human V1aR model molecules were similar binding affinity energies of -7,38±0.3 kJ/mol for OT-GKR and −11,11±0.6 kJ/mol for OT. Figure 2E and Table 1 show OT-GKR and OT docking at respective binding sites. Both molecules were docked in positions of Q104, K128, Q311, L335, S338 and N340. The OT-GKR was bound exclusively in positions of G134, S138 and A299.


Oxytocin-Gly-Lys-Arg: a novel cardiomyogenic peptide.

Danalache BA, Gutkowska J, Slusarz MJ, Berezowska I, Jankowski M - PLoS ONE (2010)

Molecular docking of 3-D models of activated human OTR and V1aR with OT/OT-GKR peptides obtained by the MolDock Optimizer algorithm from Molegro Virtual Docker.(A) The front upright view position (side view) of the OTR-OT- GKR complex structure. (A1) The section (rectangle) shown in panel A1 from the top a intracellular view (i.e. rotation by 90° out of plane) of the marked section in A, demonstrate OT-GKR (green) in active conformation inside the OT binding site (the transmembrane helices in red and the cavity in violet). (B) side view of the V1aR-OT- GKR complex, (B1) V1aR top view. (C1) detail of docking view of OTR-OT-GKR complex, and (C2) detail of V1aR-OT-GKR complex. D displays the schematic model of human OTRs with marked amino acid residues that are putatively involved in ligand-binding. The amino acid residues in black circles have been proposed as OT docking sites, and the red bars represent docking sites of OT-GKR. (E) Schematic model of human vasopressin V1aR binding with OT-GKR and OT. Amino acid residues are identified by a 1-letter code in Table 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2964328&req=5

pone-0013643-g002: Molecular docking of 3-D models of activated human OTR and V1aR with OT/OT-GKR peptides obtained by the MolDock Optimizer algorithm from Molegro Virtual Docker.(A) The front upright view position (side view) of the OTR-OT- GKR complex structure. (A1) The section (rectangle) shown in panel A1 from the top a intracellular view (i.e. rotation by 90° out of plane) of the marked section in A, demonstrate OT-GKR (green) in active conformation inside the OT binding site (the transmembrane helices in red and the cavity in violet). (B) side view of the V1aR-OT- GKR complex, (B1) V1aR top view. (C1) detail of docking view of OTR-OT-GKR complex, and (C2) detail of V1aR-OT-GKR complex. D displays the schematic model of human OTRs with marked amino acid residues that are putatively involved in ligand-binding. The amino acid residues in black circles have been proposed as OT docking sites, and the red bars represent docking sites of OT-GKR. (E) Schematic model of human vasopressin V1aR binding with OT-GKR and OT. Amino acid residues are identified by a 1-letter code in Table 1.
Mentions: Because of significant molecular differences between OT-GKR and OT, the question has been raised whether OT-GKR interacts with OTR binding sites. For this reason, we performed computational docking analysis of OT molecules in OTR binding sites. Figure 2 illustrates the docking of 3-D human OTR with OT-GKR and OT-modeled molecules in front upright view (Fig. 2A) and a view from the extracellular side (Fig. 2B). Three conformations for both OT-GKR and OT were analyzed. Six related receptor-Gα-segment-OT-GKR complexes were obtained. Possible hydrophobic and electrostatic interaction points in dynamic complexes of these molecules were indicated by estimated binding affinity energies of −6.6±0.4 kJ/mol for OT-GKR and −11.8±0.6 kJ/mol for OT. Using distance criteria, the program identified receptor amino acid residues interacting with ligands. The essential hydrogen bond and strong electrostatic interactions between both OT molecules and the receptors were characterized by visual inspection. The results are reported in Figure 2D and Table 1. Several amino acid residues have been proposed to interact with OT and OT-GKR in the OTR model where the red bars represent docking with OT-GKR, and the black bars indicate docking with OT molecule (Fig. 2D). Docking to OTR in positions V115, K116, Q119, M123, Q171, F185, T205, Y209 and Q295 was noted for both OT and OT-GKR models (Table 1). These docking positions constituted 42% of all observed docking sites of OT-GKR. Among OT-GKR interactions, the special notice should be given to binding of the arginine-12 (R12), (Fig. 2 D) to OTR. We also analyzed the docking of OT-GKR and OT molecules in the 3-D human V1aR model molecules were similar binding affinity energies of -7,38±0.3 kJ/mol for OT-GKR and −11,11±0.6 kJ/mol for OT. Figure 2E and Table 1 show OT-GKR and OT docking at respective binding sites. Both molecules were docked in positions of Q104, K128, Q311, L335, S338 and N340. The OT-GKR was bound exclusively in positions of G134, S138 and A299.

Bottom Line: In embryonic P19 cells, OT-GKR induced contracting cell colonies and ventricular CM markers more potently than OT, an effect being suppressed by OT antagonists and OTR-specific small interfering (si) RNA.The V1a receptor antagonist and specific si-RNA also significantly reduced OT-GKR-stimulated P19 contracting cells.These results raise the possibility that C-terminally extended OT molecules stimulate CM differentiation and contribute to heart growth during fetal life.

View Article: PubMed Central - PubMed

Affiliation: Research Centre, Centre Hospitalier de l'Université de Montréal - Hôtel-Dieu, Montreal, Quebec, Canada.

ABSTRACT

Background: Oxytocin (OT), synthesized in the heart, has the ability to heal injured hearts and to promote cardiomyogenesis from stem cells. Recently, we reported that the OT-GKR molecule, a processing intermediate of OT, potently increased the spontaneous formation of cardiomyocytes (CM) in embryonic stem D3 cells and augmented glucose uptake in newborn rat CM above the level stimulated by OT. In the present experiments, we investigated whether OT-GKR exists in fetal and newborn rodent hearts, interacts with the OT receptors (OTR) and primes the generation of contracting cells expressing CM markers in P19 cells, a model for the study of early heart differentiation.

Methodology/principal findings: High performance liquid chromatography of newborn rat heart extracts indicated that OT-GKR was a dominant form of OT. Immunocytochemistry of mouse embryos (embryonic day 15) showed cardiac OT-GKR accumulation and OTR expression. Computerized molecular modeling revealed OT-GKR docking to active OTR sites and to V1a receptor of vasopressin. In embryonic P19 cells, OT-GKR induced contracting cell colonies and ventricular CM markers more potently than OT, an effect being suppressed by OT antagonists and OTR-specific small interfering (si) RNA. The V1a receptor antagonist and specific si-RNA also significantly reduced OT-GKR-stimulated P19 contracting cells. In comparison to OT, OT-GKR induced in P19 cells less α-actinin, myogenin and MyoD mRNA, skeletal muscle markers.

Conclusions/significance: These results raise the possibility that C-terminally extended OT molecules stimulate CM differentiation and contribute to heart growth during fetal life.

Show MeSH