In cardiac myocytes, voltage-gated L-type calcium channels play a crucial role in excitation and contraction coupling. The two main receptor systems involved in the regulation of contractility are the muscarinic and the β-adrenergic, which exert opposite effects on the calcium current (I_Ca) through the activation of G-proteins. Recent reports have demonstrated that the different subtypes of β-adrenergic receptors are coupled to both G_i and/or G_s; β₂-AR can activate G_s and G_i proteins, whereas β₁-AR couple only to G_s (Zhu et al. 2001). The modulation of I_Ca is mainly due to hormones and mediators acting through the activation of different protein kinases; among them, phosphoinositide 3-kinases (PI3Ks) have been recently discovered to play an important role in the transduction of the intracellular signalling mediated by G-protein coupled and tyrosine kinase receptors (Macrez et al. 2001; Steinberg, 2001). A signalling pathway from PI3K to the serine/threonine protein kinase Akt/PKB is implicated in some cellular responses; in particular, it has been shown that in adult mouse cardiac myocytes, β₂-ARs coupled to G_i signalling, activate PI3K-Akt pathway through the Gβλ subunit and are directly implicated in the survival effect (Naga Prasad et al. 2000; Zhu et al. 2001).

In our study, we compared the effect of the β-adrenergic agonist, Isoproterenol (Iso), on wild-type and PI3K knock-out mice, looking at the modulation of contractility in atrial and papillary muscles and of I_Ca in isolated ventricular cells (Gallo et al. 2001). Animals were killed according to the procedures of the National guidelines.

The amplitude of contractility evoked by Iso in wild-type atrial muscle was significantly different, compared with the knock-out animals. The maximal Iso (1 mM) effect was 7.8 ± 0.7-fold with respect to the control values in n = 5 experiments in the wild-type mice (all data are expressed as means ± S.E.M.). The positive inotropic effect was significantly reduced in the knock-out mice (4.5 ± 0.6; n = 6, t test**). Surprisingly, in the papillary muscle the inotropic effect seemed rather to be higher in the PI3K-deficient mice (6.3 ± 2.9; n = 5), than the wild-type (3.4 ± 0.5; n = 6). The role of PI3Kλ in the modulation of I_Ca by Iso was studied in adult cardiac myocytes, enzymatically dissociated from wild-type and knock-out animals. In 13 cells from wild-type mice, the percentage of stimulation of Iso above the control values was 30.2 ± 2.0 %, and, similarly to papillary muscle, the Iso effect was higher in the knock-out cells (86.2 ± 6.5 %, n = 5, t test**). Taken together, these data support the idea that, in cardiac muscle, PI3Kλ has a physiological role in the regulation of I_Ca and contractility. The differential effects of Iso observed in atrial and ventricular preparations suggest that in ventricular cells, the intracellular kinase-mediated signal, which modulates I_Ca and inotropism, should be linked to a G_i β-ARs-coupled subtype. The opposite inotropic response of atrial and ventricular tissues to Iso might be explained on the basis of different β-ARs distribution within the heart.

All procedures accord with current National guidelines.