Studio del ruolo della fosfodiesterasi 5 nella modulazione della frequenza di contrazione

Compartmentalization of cAMP and PKA activity in cardiac muscle cells plays a key role in maintaining basal and enhanced contractility stimulated by sympathetic nerve activity. In cardiomyocytes, activation of adrenergic receptor increases cAMP synthesis, which is countered by the hydrolytic activity of selective phosphodiesterases (PDEs). Evidences have been accumulated that PDE5 protein is expressed in cardiomyocytes and up-regulated in several pathological conditions (Nagendran J et al.2007;Takimoto E. et al.2005). Pharmacological inhibition of PDE5 with sildenafil, a selective and potent inhibitor, has been shown effective in counteracting cardiac hypertrophy in animal models. Encouraging data are also emerging from clinical trials in heart failure (Guazzi M et al. 2011; Andersen A et al. 2013) and in diabetic cardiomyopathy exerting anti-remodelling effects (Giannetta E. et al 2015). However, the molecular mechanisms of sildenafil action on cardiac homeostasis are still under investigation. It is known that sildenafil, occupying the catalytic site as pseudo substrates, blocks the esterase activity and leads to increased cGMP levels. Rise of intra-myocyte cGMP subsequently modulates the activation of PKG. (Francis SH et al 2011). The negative inotropic effect of PDE5 inhibitors after -adrenergic-stimulation has been also reported in several species (Lee DI et al. 2010; Senzaki H et al 2001; Borlaug BA et al. 2005) including humans. However, the effects on the frequency of contraction have not been previously addressed because of the intrinsic limitation of the investigated models. To examine the contribution of PDE5A inhibition to 1AR-, 2AR- and 3AR-stimulated contraction rate, we used in vitro beating assay performed on cultured neonatal cardiomyocytes and we measured the cardiac rate under different conditions. Contrary to adult, the neonatal ventriculocytes maintain their plasticity allowing them to form spontaneous beating syncytia and to respond to -adrenergic stimuli. Using a combined pharmacological and genetic approach based on selective disruption of the activities and genes encoding different ARs and PDEs.