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effects of VSs were independent from endocardial endothelium (EE) and nitric
oxide–cGMP mechanism (Corti et al. 2004 ), in the eel heart the VS-1-mediated
negative inotropism required the presence of an intact EE and the activation of
NO-cGMP-PKG pathway (Imbrogno et al. 2004 ). These findings provided the first
evidence that vasostatins exert cardiotropic action in amphibian and fish heart, thus
suggesting their long evolutionary history, as well as their species-specific mecha-
nisms of action. Further studies on isolated working eel and frog heart preparations
have been performed to study the role of the cytoskeleton in the VSs-mediated ino-
tropic response (Mazza et al. 2007 ). In both eel and frog hearts, VSs-mediated-
negative inotropy was abolished by treatment with inhibitors of cytoskeleton
reorganization, such as cytochalasin-D, suggesting that changes in cytoskeletal
dynamics play a crucial role in the negative inotropic influence of VSs on these
preparations.
The negative inotropic and lusitropic effects of VSs have been further demon-
strated in ex-vivo studies on the isolated rat heart. In particular, VS-1 and VS-1-
derived peptides containing the disulfide bridged loop reduced cardiac contractility,
both under basal conditions and after β-adrenergic stimulation (Cerra et al. 2006 ,
2008 ). The action of VS-1 involved both Gi/o protein, as suggested by the blocking
effect of pertussis toxin (PTX), and NO-cGMP-PKG pathway (Cerra et al. 2008 ).
Moreover, it has been shown that, like in the eel and frog heart (Mazza et al. 2007 ),
cytoskeleton integrity is involved in the modulation of contractility exerted by
human recombinant Vasostatin-1 (hrVS-1) and rat chromogranin A 1–64 (rCgA1–
64) in the rat heart (Angelone et al. 2010 ). Indeed, cytoskeleton impairment by
either cytochalasin-D, Butanedione monoxime (BDM), wortmannin or W-7 abol-
ished VSs-induced inotropic response. Moreover, hrVS-1 stimulated actin polymer-
ization in rat cardiac H9C2 cells, supporting the hypothesis that the actin cytoskeletal
network strongly contributes to the cardiotropic action of CgA-derived peptides.
The fact that in the isolated rat heart the negative inotropic effect of VSs was not
accompanied by significant alterations in heart rate and coronary resistances,
strongly suggested a direct action of these peptides. In vitro studies on the isolated
rat papillary muscle confirmed the ability of VSs to exert direct inotropic effects.
Indeed, the isolated papillary muscle is driven at constant frequency and perfused at
constant flow, to avoid any possible alteration of contractility due to variations of
heart rate and coronary flow. In these experimental conditions, VS-1 induced dose
and time-dependent effects, under both basal conditions and after β-adrenergic
stimulation (Gallo et al. 2007 ).
In agreement with the results obtained on the isolated rat heart, hrVS-1 reduced
in a concentration-dependent (5–100 nM) manner the inotropic effect of Iso in
papillary muscle. The minimal effective concentration of hrVS-1 was 5 nM, while
the higher concentration of hrVS-1 reduced to about 70% the inotropic response to
β-adrenergic stimulation (Gallo et al. 2007 ).
To investigate the structure-function relationship of different VSs, the effects of
two modified peptides, i.e., rCgA1–64 without the S-S bridge (rCgA1–64SH) and
rCgA1–64 oxidized (rCgA1– 64OX), were compared to those exerted by N-terminal
fragment of CgA, reproducing the native rat sequence (rCgA1–64 with S-S bridge:
G. Alloatti and M.P. Gallo