Derangement of sarcoplasmic reticulum (SR) function play a primary role in the pathogenesis of cardiac contractile deficit in heart failure (HF). The PI3K/AKT is a complex signalling pathway with a central role in the modulation of both cardiac function and structure; an imbalance in its components may contribute to contractile dysfunction and myocardial structural remodelling in HF. In particular, the cardiac contractility modulation, in which the PI3K/Akt pathway is involved, is mainly regulated by β-adrenergic signaling. The pleckstrin-homology (PH) domain is a highly conserved protein domain, involved in PI3K-mediated membrane recruitment, and subsequent activation, of signaling pathways, including Akt. The diverse effects mediated by PI3K/Akt signalling in the heart clearly support an important biological and pathophysiological role for this signalling cascade. The PI3K/Akt signalling pathways are involved in a wide variety of diseases including myocardial hypertrophy and contractility, heart failure, and preconditioning. The aim of this study was to further investigate the role of PI3K/Akt pathway on the modulation of the cardiac excitation-contraction coupling (ECC). We focused our attention on Akt, because the role of this kinase results still unclear. To pursue this purpose we inhibited Akt through two experimental approaches: first, we used two chemical unrelated compounds (Compound A and B) able to interact with kineases PH-domain to prevent the recognition of the PIP3 inositol ring and subsequent activation; second, Akt gene silencing through RNA intereference technique. With these two methods, we were able to rule out compound-dependent effects due to their potential aspecificity and to assert the effective consequence of Akt activity ablation on the modulation of cardiac function. PH-antagonism (PH-antag) increased baseline contractility, an effect inhibited by β1+β2-AR blockade. The effect of PH-antag was larger in the presence of ISO, abolished by β1+β2-AR blockade, but only partially reduced by selective blockade of either β1- or β2-AR. Basal twitch amplitude were increased in Akt1 silenced cells; both compounds did not affect twitch amplitude on transfected myocytes. Both PH-domain inhibitors stimulated SR function (NCX blockade) increasing the SR reloading steepness, the Ca2+ reuptake rate and the EC-coupling gain. Also increase calcium leakage without affecting SR content. So we can conclude that PH-antag unmask the inhibitory effect of Akt acting trough SR system increasing SERCA and RyR activity.

(2010). Akt-Pka interaction in the modulation of cardiac excitation - contraction coupling. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).

Akt-Pka interaction in the modulation of cardiac excitation - contraction coupling

CHISCI, RICCARDO
2010-01-08

Abstract

Derangement of sarcoplasmic reticulum (SR) function play a primary role in the pathogenesis of cardiac contractile deficit in heart failure (HF). The PI3K/AKT is a complex signalling pathway with a central role in the modulation of both cardiac function and structure; an imbalance in its components may contribute to contractile dysfunction and myocardial structural remodelling in HF. In particular, the cardiac contractility modulation, in which the PI3K/Akt pathway is involved, is mainly regulated by β-adrenergic signaling. The pleckstrin-homology (PH) domain is a highly conserved protein domain, involved in PI3K-mediated membrane recruitment, and subsequent activation, of signaling pathways, including Akt. The diverse effects mediated by PI3K/Akt signalling in the heart clearly support an important biological and pathophysiological role for this signalling cascade. The PI3K/Akt signalling pathways are involved in a wide variety of diseases including myocardial hypertrophy and contractility, heart failure, and preconditioning. The aim of this study was to further investigate the role of PI3K/Akt pathway on the modulation of the cardiac excitation-contraction coupling (ECC). We focused our attention on Akt, because the role of this kinase results still unclear. To pursue this purpose we inhibited Akt through two experimental approaches: first, we used two chemical unrelated compounds (Compound A and B) able to interact with kineases PH-domain to prevent the recognition of the PIP3 inositol ring and subsequent activation; second, Akt gene silencing through RNA intereference technique. With these two methods, we were able to rule out compound-dependent effects due to their potential aspecificity and to assert the effective consequence of Akt activity ablation on the modulation of cardiac function. PH-antagonism (PH-antag) increased baseline contractility, an effect inhibited by β1+β2-AR blockade. The effect of PH-antag was larger in the presence of ISO, abolished by β1+β2-AR blockade, but only partially reduced by selective blockade of either β1- or β2-AR. Basal twitch amplitude were increased in Akt1 silenced cells; both compounds did not affect twitch amplitude on transfected myocytes. Both PH-domain inhibitors stimulated SR function (NCX blockade) increasing the SR reloading steepness, the Ca2+ reuptake rate and the EC-coupling gain. Also increase calcium leakage without affecting SR content. So we can conclude that PH-antag unmask the inhibitory effect of Akt acting trough SR system increasing SERCA and RyR activity.
ROCCHETTI, MARCELLA
Akt; PKA; Excitation-Contraction coupling; PH-Domain Inhibitor
BIO/09 - FISIOLOGIA
English
Scuola di dottorato di Scienze
BIOLOGIA - 48R
22
2008/2009
(2010). Akt-Pka interaction in the modulation of cardiac excitation - contraction coupling. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/7484
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