Functional activity of the carboxyl-terminally extended oxytocin precursor Peptide during cardiac differentiation of embryonic stem cells.
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.
Gassanov
Natig
N
Department of Internal Medicine III, University of Cologne, Cologne, Germany.
Devost
Dominic
D
Danalache
Bogdan
B
Noiseux
Nicolas
N
Jankowski
Marek
M
Zingg
Hans H
HH
Gutkowska
Jolanta
J
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006321:Heart / Q000196:embryology*; D007150:Immunohistochemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D010121:Oxytocin / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D010455:Peptides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D010121:Oxytocin