Phosphatidylinositol 3-kinase offsets cAMP-mediated positive inotropic effect via inhibiting Ca²⁺ influx in cardiomyocytes

Phosphoinositide 3-kinase (PI3K) has been implicated in β₂-adrenergic receptor (β₂-AR)/G _i-mediated compartmentation of the concurrent G_s-cAMP signaling, negating β₂-AR-induced phospholamban phosphorylation and the positive inotropic and lusitropic responses in cardiomyocytes. However, it is unclear whether PI3K crosstalks with the β₁-AR signal transduction, and even more generally, with the cAMP/PKA pathway. In this study, we show that selective β₁-AR stimulation markedly increases PI3K activity in adult rat cardiomyocytes. Inhibition of PI3K by LY294002 significantly enhances β₁-AR-induced increases in L-type Ca ²⁺ currents, intracellular Ca²⁺ transients, and myocyte contractility, without altering the receptor-mediated phosphorylation of phospholamban. The LY294002 potentiating effects are completely prevented by βARK-ct, a peptide inhibitor of β-adrenergic receptor kinase-1 (βARK1) as well as G_βγ signaling, but not by disrupting G_i function with pertussis toxin. Moreover, forskolin, an adenylyl cyclase activator, also elevates PI3K activity and inhibition of PI3K enhances forskolin-induced contractile response in a βARK-ct sensitive manner. In contrast, PI3K inhibition affects neither the basal contractility nor high extracellular Ca²⁺-induced increase in myocyte contraction. These results suggest that β₁-AR stimulation activates PI3K via a PKA-dependent mechanism, and that G_βγ and the subsequent activation of βARK1 are critically involved in the PKA-induced PI3K signaling which, in turn, negates cAMP-induced positive inotropic effect via inhibiting sarcolemmal Ca²⁺ influx and the subsequent increase in intracellular Ca²⁺ transients, without altering the receptor-mediated phospholamban phosphorylation, in intact cardiomyocytes.