Abstract Body: Phosphoinositide 3-kinase (PI3Kγ) null mice (PI3Kγ^-/-) show increased ventricular rate/contraction. However, it is unknown whether PI3Kγ regulates calcium recycling machinery underlying this phenotype. Primary adult cardiomyocytes isolated from WT, PI3Kγ^-/- mice and mice with cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ_inact) in PI3Kγ^-/- background (PI3Kγ_inact/PI3Kγ^-/-) were measured for contractility, and basal and caffeine induced calcium cycling. Phosphorylation status of proteins involved in calcium cycling like Ryanodine Receptor (RyR2), sarcoendoplasmic reticulum (SR) calcium ATPase 2a (SERCA2) and phospholamban (PLN) assessed. SERCA2 interaction with PLN evaluated in the presence or absence of PI3Kγ. Purified proteins were used to evaluate protein-protein interaction between PLN regulator Ca²⁺/Calmodulin dependent protein kinase (CaMKII) and PI3Kγ. Basal and caffeine mediated calcium re-uptake by SR were reduced in primary adult cardiomyocytes isolated from PI3Kγ^-/- mice. PI3Kγ^-/- cardiomyocytes showed significant reduction in phosphorylation of PLN at Thr17, a key regulator of SR calcium re-uptake without changes in phosphorylation at Ser16. Furthermore, this loss in PLN phosphorylation in PI3Kγ^-/-cardiomyocytes resulted in augmented PLN-SERCA2 interaction. Surprisingly, basal and caffeine-induced calcium re-uptake and PLN phosphorylation at Thr17 were normalized in PI3Kγ_inact/PI3Kγ^-/- mice. This results in decreased PLN-SERCA2 interaction that preserves SR calcium re-uptake reflecting kinase-independent function of PI3Kγ in regulation of SR calcium load and PLN phosphorylation. Co-immunoprecipitation studies showed that PI3Kγ interacts with CaMKII regulating CaMKII recruitment to PLN through its kinase-independent scaffolding function that determines PLN phosphorylation. Furthermore, surface plasmon resonance studies showed that purified PI3Kγ protein robustly interacts with purified CaMKII protein supporting kinase-independent scaffolding function of PI3Kγ.