Functional Activity Of The Carboxy-Terminally Extended Oxytocin Precursor Peptide During Cardiac Differentiation Of Embryonic Stem Cells

¹ Department of Internal Medicine III, University of Cologne, Germany; Research Centre, Centre hospitalier de l'Université de Montréal (CHUM)- Hôtel-Dieu, 3850 Saint Urbain, Montreal (QC) H2W 1T7, Canada
² Department of Pharmacology and Therapeutics, McGill University, 3655, Sir William Osler Promenade, Montreal (QC) H3G 1Y6, Canada
³ Research Centre, Centre hospitalier de l'Université de Montréal (CHUM)- Hôtel-Dieu, 3850 Saint Urbain, Montreal (QC) H2W 1T7, Canada

^* To whom correspondence should be addressed. E-mail: jolanta.gutkowska{at}umontreal.ca.

	Abstract

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) application. To obtain further insights into the mechanisms of OT-GKR-induced cardiac effects, we generated ES cell lines overexpressing 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 MLC-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 Cx43 protein in OT-GKR/EGFP-expressing cells further substantiated the evidence that OT-GKR modifies cardiac differentiation towards the ventricular sublineage. In conclusion, this report provides new evidence of the biological activity of OT-X, notably OT-GKR, during cardiomyogenic differentiation.

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