Abstract-Ca2ϩ transients measured in failing human ventricular myocytes exhibit reduced amplitude, slowed relaxation, and blunted frequency dependence. In the companion article (O'Rourke B, Kass DA, Tomaselli GF, Kääb S, Tunin R, Marbán E. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart, I: experimental studies. Circ Res. 1999;84:562-570 Key Words: excitation-contraction coupling Ⅲ heart failure Ⅲ midmyocardial ventricular action potential Ⅲ Ca 2ϩ transient R ecent studies using the canine tachycardia pacinginduced model of heart failure 1-8 demonstrate that changes in cellular electrophysiological and excitationcontraction (E-C) coupling processes are qualitatively similar to those observed in cells isolated from failing human heart. In human heart failure, I K1 current density measured at hyperpolarized membrane potentials is reduced by Ϸ50%, 9,10 and density of the transient outward current I to1 is reduced by Ϸ75% in subepicardial 11 and Ϸ40% in midmyocardial ventricular cells 9 and is unchanged in subendocardial ventricular cells. 11 The magnitude of I K1 is reduced by Ϸ40%, and that of I to1 by Ϸ70% in failing canine midmyocardial cells. 5 Expression of proteins involved in E-C coupling is also altered in human heart failure. Sarcoplasmic reticulum (SR) Ca 2ϩ ATPase mRNA level, 12-16 protein level, 12,17,18 and uptake rate 19 are reduced by Ϸ50% in end-stage heart failure. Na ϩ /Ca 2ϩ exchanger (NCX) mRNA levels are increased by Ϸ55% to 79%, 12,20 and NCX protein levels increase 36% to 160%. 12,20 -22

Ca transients measured in failing human ventricular myocytes exhibit reduced amplitude and slowed relaxation [Beuckelmann, D.J., Nabauer, M., Erdmann, E., 1992. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85, 1046-1055; Gwathmey, J.K., Copelas, L., MacKinnon, R., Schoen, F.J., Feldman, M.D., Grossman, W., Morgan, J.P., 1987. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ. Res. 61, 70-76; Kaab, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P.H., Kass, D.A., Marban, E., Tomaselli, G.F., 1996. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ. Res. 78(2); Li, H.G., Jones, D.L., Yee, R., Klein, G.J., 1992. Electrophysiologic substrate associated with pacing-induced hert failure in dogs: potential value of programmed stimulation in predicting sudden death. J. Am. Coll. Cardiol. 19(2), 444-449; Vermeulen, J.T., McGuire, M.A., Opthof, T., Colonel, R., Bakker, J.M.T.d., Klopping, C., Janse, M.J., 1994. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc. Res. 28, 1547-1554.] and blunted frequency dependence [Davies, C.H., Davia, K., Bennett, J.G., Pepper, J.R., Poole-Wilson, P.A., Harding, S.E., 1995. Reduced contraction and altered frequency response of isolated ventricular myocytes from patients with heart failure. Circulation, 92, 2540-2549; Hasenfuss, G., Reinecke, H., Studer, R., Meyer, M., Pieske, B., Holtz, J., Holubarsch, C., Posival, H., Just, H., Drexler, H., 1994. Relation between myocardial function and expression of sarcoplasmic reticulum Ca-ATPase in failing and nonfailing human myocardium. Circ. Res. 75, 434-442; Hasenfuss, G., Reinecke, H., Studer, R., Pieske, B., Meyer, M., Drexler, H., Just, H., 1996. Calcium cycling proteins and force-frequency relationships in heart failure. Basic Res. Cardiol. 91, 17-22; Monte, F.D., O'Gara, P., Poole-Wilson, P.A., Yacoub, M., Harding, S.E., 1995. Cell geometry and contractile abnormalities of myocytes from failing human left ventricle.

Urotensin-II (U-II) is a vasoactive peptide with many effects in the cardiovascular system. It has been described to induce vascular smooth muscle cell (SMC) proliferation, and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling; however, its signalling pathway still remains unclear. The aim of this study was to elucidate the role of calcium (Ca 2þ)-dependent signalling and alternative signalling pathways in UIIevoked VSMCs proliferation, focussing our attention on store-operated Ca 2þ entry (SOCE) pathway and epithelium growth factor receptor (EGFR) transactivation. We used 5-bromo-2-deoxyuridine (BrdU) labeling, molecular knockdown with small interfering RNA (siRNA) and immunofluorescence to study UIIpromoted aortic SMC proliferation. Ca 2þ mobilization assays were performed to study Ca 2þ entry in cultured SMC. We found that UII enhanced intracellular Ca 2þ concentration ([Ca 2þ ]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as STIM1, Orai1, or TRPC1. Moreover, UII stimulated SMC proliferation and Ca 2þ /cAMP response elementbinding protein (CREB) activation through SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Additionally, we determined that epithelium growth factor receptor (EGFR) transactivation, extracellular signal-regulated kinase (ERK) and Ca 2þ /calmodulin-dependent kinase (CaMK) signaling pathways were involved in UII-mediated Ca 2þ influx, CREB activation and VSMCs proliferation. Our data show for the first time that UII-induced SMC proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1 and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.