1573 Endothelial-Mediated Effects of VS-1 and CST
To investigate the possible role of endothelial-derived mediators in the action of
CgA-derived peptides, the effects of VSs and CST were initially tested on the frog
heart. In contrast with the mammalian heart, in which both coronary vascular and
endocardial endothelium are present, in the avascular frog heart the endocardial
endothelium represents the only barrier between blood and cardiac cells. The fact
that both VSs (Corti et al. 2002 ) and CST (Mazza et al. 2008 ) maintained their abil-
ity to reduce cardiac contractility also in the frog heart, firstly suggested a direct
cardiac action of these peptides.
Studies performed on the isolated rat heart confirmed the involvement of the
vascular endothelium in CST-induced negative inotropism also in mammals
(Angelone et al. 2012b). CST-induced negative inotropism and lusitropism involved
β2/β3-adrenergic receptors (AR). In particular, CST interaction with β2-AR acti-
vated PI3K/eNOS pathway, increased cGMP levels, and induced activation of type
2 phosphodiesterases (PDE2), leading to a decrease of cAMP levels. The abrogation
of CST-dependent negative effect following functional denudation of the endothe-
lium with Triton X-100 strongly suggested that the action of CST is due to stimula-
tion of the vascular endothelium. This assumption was further confirmed by the
ability of CST to stimulate eNOS phosphorylation in both cardiac tissue and cul-
tured human umbilical vein endothelial cells. CST also increased S-nitrosylation of
both phospholamban and β-arrestin in ventricular extracts. Taken together, these
data suggest that endothelium derived NO, PDE2 and S-nitrosylation play crucial
roles in the CST regulation of cardiac function (Angelone et al. 2012b).
To test the involvement of NO released from endocardial endothelial cells, rat
papillary muscles were treated with VS-1 or CST after endothelium has been
removed with Triton X-100. While this protocol did not modify the inotropic effect
induced by Iso, the anti-adrenergic effect of the two CgA-derived peptides was
completely abolished, suggesting the endothelial origin of NO (Bassino et al. 2011 ;
Gallo et al. 2007 ). Studies from our laboratory provided novel information on the
mechanisms of the cardiac antiadrenergic action of CgA-derived peptides, high-
lighting the crucial involvement of a Ca2+-independent/PI3K-dependent NO release
from endothelial cells. To characterize the mechanisms responsible for the anti-
adrenergic effect of VS-1 and CST, we studied the effect of these peptides on L-type
calcium current (ICa,L) or Ca2+ transients in isolated rat ventricular cardiomyocytes.
In agreement with the results obtained in isolated papillary muscles treated with
Triton X-100, L-type calcium current or Ca2+ transients measurements confirmed
that, in the absence of endothelial cells, VS-1 and CST are ineffective both in basal
conditions and after Iso stimulation (Bassino et al. 2011 ; Gallo et al. 2007 ). The lack
of effect of VS-1 and CST on basal and Iso-stimulated Ca2+ transients amplitude
represents an indirect demonstration that the ionic currents involved in the action
potential are not affected by these peptides. In addition, the lack of any inhibitory
effect of the two CgA-derived peptides on isolated ventricular cells confirmed that,
at least in physiological conditions and with acute stimulation by the peptides, they
Signalling Pathways of CgA-Derived Peptides in Cardiac and endothelial cells