A Specific Role of Phosphatidylinositol 3–Kinase γ: A Regulation of Autonomic Ca(2)+ Oscillations in Cardiac Cells

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase–dependent pathway that stimulates PLCγ and generates IP(3), a breakdown product of phosphatidylinositol 4,5–bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca(2+) spiking). PIP2 is phosphorylated on 3′ by phosphoinositide 3–kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5–trisphosphate, regulates activity of PLCγ. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCγ, Ca(2+) homeostasis. However, although PI3Kα and -β have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kγ. We report that neonatal rat cardiac cells in culture express PI3Kα, -β, and -γ. The purinergic agonist predominantly activates PI3Kγ. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCγ as well as IP(3) generation in ATP-stimulated cells. Furthermore, an anti-PI3Kγ, but not an anti-PI3Kβ, injected in the cells prevents the effect of ATP on cell Ca(2+) spiking. A dominant negative mutant of PI3Kγ transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca(2+) spiking of wild-type but not of PI3Kγ(2/2) embryonic stem cell–derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCγ. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kγ antibody and a dominant negative mutant of PI3Kγ injected or transfected into cells prevent the latter event. We conclude that PI3Kγ activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kγ to fully activate PLCγ in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP(3) generation and thus cardiac autonomic activity.