Data was then exported to MS Excel and graphed using Origin 7. 0 and Sigmaplot 11. 0. For statistical analyses, average i from 25 40 cells within a microscopic field were obtained during the control period of 1 5 min from each of 5 separate HL 1 cell preparations. These averages were then compiled to obtain average control values, and comparisons were made on data col lected similarly from the same microscopic fields 15 min utes after experimental additions. Statistical differences between control and experimental values were estab lished at p 0. 05. Solutions and chemicals Standard external salt solution contained NaCl 150, KCl 6, MgCl2 1, CaCl2 1. 5, N 2 Hydroxyethylpiperazine N 2 ethanesulfonic acid 10, glucose 10. Pipette solution contained potassium aspartate 120, Na2GTP 0.

4, Na2ATP 5, MgCl2 1, EGTA 5, CaCl2 0. 1, HEPES 10. For whole cell voltage clamp measurements of membrane Ca2 currents external NaCl was substituted with n methy D gluamine chloride 150, and CaCl2 was increased to 5 to maximize Ca2 current. All solution constituents were obtained from Sigma/Aldrich, St. Louis, MO. LY 294002 was obtained from Alomone Labs, LTD, Je rusalem, Israel. The PI3 kinase isoform inhibitors PI3 kinase inhibitor 2, TGX 221 B inhibitor, AS 252424 isoform inhibitor. and the AKT inhibitor, Triciribine were obtained from Cayman Chemical, Ann Arbor, MI. The dosages selected for the various inhibitors were based on the literature and the manufacturers instruc tions. All inhibitors were dissolved in DMSO in stock solutions and then diluted to final concentration.

The highest final concentration of DMSO by this approach was 0. 24% DMSO. Results Pharmacologic inhibition of PI3K significantly reduces i, and Ca2 transients in HL 1 cardiomyocytes Action potentials and corresponding spontaneous transi ents in intracellular Ca2 , i, occur in approximately 40% of non confluent immortalized mouse HL 1 cardio myocytes. Synchronous Ca2 transients in three such cells are shown in Figure 1A. Perfusing the cells with LY 294002, a potent inhibitor of phosphoinositde 3 kinases, inhibited Ca2 tran sients within 2 minutes, and this effect was partially reversed on washout. When all cells within a micro scopic field, i. e. those showing Ca2 transients and those without Brefeldin_A transients, were included in the com putation of mean i, the Ca2 transients again were evident, but the averaging reduced their magnitude, Figure 1B.

LY 294002 again abolished the Ca2 transients and diminished total i, Figure 1B. Washout restored total i, but the Ca2 transients were no longer apparent, except for partial restoration in 3 cells out of the 10 of 37 cells showing Ca2 transients. LY 294002 at 1 uM also inhibited Ca2 tran sients with some restoration on washout, Figure 1C. LY 294002 at 1 uM also significantly reduced total i, Table 1, with modest but insignificant reversal on wash out within 5 minutes, Figure 1D.