© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Investigation of signaling pathways that mediate the inotropic effect of urotensin-II in human heart
Department of Medicine, University of Queensland, The Prince Charles Hospital, Rode Road Chermside, 4032 Queensland, Australia
* Corresponding author. Tel.: +61-7-3350-8792; fax: +61-7-3359-2173. Email address: russell{at}medicine.uq.edu.au
Objective: This study investigated signaling pathways that may contribute to the potent positive inotropic effect of human urotensin-II (hU-II) in human isolated right atrial trabeculae obtained from patients with coronary artery disease. Methods: Trabeculae were set up in tissue baths and stimulated to contract at 1 Hz. Tissues were incubated with 20 nM hU-II with or without phorbol 12-myristate 13-acetate (PMA, 10 µM) to desensitize PKC, the PKC inhibitor chelerythrine (10 µM), 10 µM 4
-phorbol that does not desensitize PKC, the myosin light chain kinase inhibitor wortmannin (50 nM, 10 µM), or the Rho kinase inhibitor Y-27632 (0.1–10 µM). Activated RhoA was determined by affinity immunoprecipitation, and phosphorylation of signaling proteins was determined by SDS-PAGE. Results: hU-II caused a potent positive inotropic response in atrial trabeculae, and this was concomitant with increased phosphorylation of regulatory myosin light chain (MLC-2, 1.8±0.4-fold, P<0.05, n=6) and PKC/βII (1.4±0.2-fold compared to non-stimulated controls, P<0.05, n=7). Pretreatment of tissues with PMA caused a marked reduction in the inotropic effect of hU-II, but did not affect hU-II-mediated phosphorylation of MLC-2. The inotropic response was inhibited by chelerythrine, but not 4-phorbol or wortmannin. Although Y-27632 also reduced the positive inotropic response to hU-II, this was associated with a marked reduction in basal force of contraction. RhoA.GTP was immunoprecipitated in tissues pretreated with or without hU-II, with findings showing no detectable activation of RhoA in the agonist stimulated tissues. Conclusions: The findings indicated that hU-II increased force of contraction in human heart via a PKC-dependent mechanism and increased phosphorylation of MLC-2, although this was independent of PKC. The positive inotropic effect was independent of myosin light chain kinase and RhoA-Rho kinase signaling pathways.
KEYWORDS Urotensin-II; Contractile function; Protein kinase C; Signal transduction