BACKGROUND: Emerging evidence suggests that phosphoinositide 3-kinase (PI3K) may modulate cardiac inotropy; however, the underlying mechanism remains elusive. We hypothesized that β2-adrenergic receptor (AR)-coupled PI3K constrains increases in cardiac inotropy through cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) activation. METHODS: We tested the effects of PI3K and PDE4 inhibition on myocardial contractility by using isolated murine cardiac myocytes to study physiologic functions (sarcomere shortening [SS] and intracellular Ca+ transients), as well as cAMP and PDE activity. RESULTS: PI3K inhibition with the reversible inhibitor LY294002 (LY) resulted in a significant increase in SS and Ca2+ handling, indicating enhanced contractility. This response depended on Giα protein activity, because incubation with pertussis toxin (an irreversible Giα inhibitor) abolished the LY-induced hypercontractility. In addition, PI3K inhibition had no greater effect on SS than both a PDE3,4 inhibitor (milrinone) and LY combined. Furthermore, LY decreased PDE4 activity in a concentration-dependent manner (58.0% of PDE4 activity at LY concentrations of 10 μM). Notably, PI3K γ coimmunoprecipitated with PDE4D. The β2-AR inverse agonist, ICI 118,551 (ICI), abolished induced increases in contractility. CONCLUSIONS: PI3K modulates myocardial contractility by a cAMP-dependent mechanism through the regulation of the catalytic activity of PDE4. Furthermore, basal agonist-independent activity of the β2-AR and its resultant cAMP production and enhancement of the catalytic activity of PDE4 through PI3K represents an example of integrative cellular signaling, which controls cAMP dynamics and thereby contractility in the cardiac myocyte. These results help to explain the mechanism by which milrinone is able to increase myocardial contractility in the absence of direct β-adrenergic stimulation and why it can further augment contractility in the presence of maximal β-adrenergic stimulation. Copyright © 2010 International Anesthesia Research Society.