Oscillations in Cardiac Cells

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase–dependent pathway that stimulates PLC g 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 1 spiking). PIP2 is phosphorylated on 3 9 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 g . PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLC g , Ca 2 1 homeostasis. However, although PI3K a and b have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3K g . We report that neonatal rat cardiac cells in culture express PI3K a , b , and g . The purinergic agonist predominantly activates PI3K g . Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLC g as well as IP 3 generation in ATP-stimulated cells. Furthermore, an anti-PI3K g , but not an anti-PI3K b , injected in the cells prevents the effect of ATP on cell Ca 2 1 spiking. A dominant negative mutant of PI3K g transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca 2 1 spiking of wild-type but not of PI3K g 2/2 embryonic stem cell–derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLC g . A dominant negative mutant of Tec blocks the purinergic effect on cell Ca 2 1 spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3K g antibody and a dominant negative mutant of PI3K g injected or transfected into cells prevent the latter event. We conclude that PI3K g activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca 2 1 spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3K g to fully activate PLC g 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.