A specific role of phosphatidylinositol 3-kinase γ:: A regulation of autonomic Ca2+ oscillations in cardiac cells

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase-dependent pathway that stimulates PKC gamma and generates IP3, a breakdown product of phosphatidylinositol 4,5-bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca2+ 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 PLC gamma. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLC gamma, Ca2+ homeostasis. However, although PI3K alpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3K gamma. We report that neonatal rat cardiac cells in culture express PI3K alpha -beta, and -gamma. The purinergic agonist predominantly activates PI3K gamma. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLC gamma as well as IP3 generation in ATP-stimulated cells. Furthermore, an anti-PI3K gamma, but not an anti-PI3K beta, injected in the cells prevents the effect of ATP on cell Ca2+ spiking. A dominant negative mutant of PI3K gamma transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca2+ spiking of wild-type but not of PI3K gamma (-/-) embryonic stem cell-derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLC gamma. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca2+ spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3K gamma antibody and a dominant negative mutant of PI3K gamma injected or transfected into cells prevent the latter event. We conclude that PI3K gamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca2+ spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3K gamma to fully activate PLC gamma in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP3 generation and thus cardiac autonomic activity.