Original Article

A Specific Role of Phosphatidylinositol 3–Kinase : A Regulation of Autonomic Ca²+ Oscillations in Cardiac Cells

Correspondence to: Michel Pucéat, Centre de Recherches de Biochimie Macromoléculaire, CNRS UPR1086, 1919 route de Mende, 34293 Montpellier, France. Tel:33-4-6761-3350 Fax:33-4-6752-1559 E-mail:puceat{at}crbm.cnrs-mop.fr.

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase–dependent pathway that stimulates PLC 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. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLC, Ca²⁺ homeostasis. However, although PI3K and -ß have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3K.

We report that neonatal rat cardiac cells in culture express PI3K, -ß, and -. The purinergic agonist predominantly activates PI3K. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLC as well as IP₃ generation in ATP-stimulated cells. Furthermore, an anti-PI3K, but not an anti-PI3Kß, injected in the cells prevents the effect of ATP on cell Ca²⁺ spiking. A dominant negative mutant of PI3K 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^2/2 embryonic stem cell–derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLC. 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 antibody and a dominant negative mutant of PI3K injected or transfected into cells prevent the latter event.

We conclude that PI3K 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 to fully activate PLC in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP₃ generation and thus cardiac autonomic activity.

Key Words: phosphoinositide kinase, calcium, tyrosine kinase, heart, automaticity

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