Phosphoinositide 3-kinase regulates excitation-contraction coupling in neonatal cardiomyocytes

The phosphoinositide 3- kinase ( PI3K) inhibitor LY- 294002 decreased steady- state contraction in neonatal rat ventricular myocytes ( NRVM). To determine whether the effect on steady- state contraction could be due to decreased intracellular Ca2+ content, Ca2+ content was assessed with fluorescent plate reader analysis by using the caffeine- releasable Ca2+ stores as an index of sarcoplasmic reticulum ( SR) Ca2+ content. Caffeine- releasable Ca2+ content was diminished in a dose- dependent manner with LY- 294002, suggesting that the decrease in steady- state contraction was due to diminished intracellular Ca2+ content. Activation of the L- type Ca2+ channel by BAY K 8644 was attenuated by LY- 294002, suggesting the effect of LY-294002 is to reduce Ca2+ influx at this channel. To investigate whether additional proteins involved in excitation- contraction ( EC) coupling are likewise regulated by PI3K activity, the effects of compounds acting at sarco( endo) plasmic reticulum Ca2+- ATPase ( SERCA2a), the ryanodine receptor, and the Na/ Ca exchanger ( NCX) were compared with LY- 294002. Inhibition of SERCA2a by thapsigargin increased basal Ca2+ levels in contrast to LY- 294002, indicating that SERCA2a activity is sustained in the presence of LY- 294002. Ryanodine decreased SR Ca2+ content. The additive effect with coadministration of LY- 294002 could be attributed to a decrease in Ca2+ influx at the L- type Ca2+ channel. The NCX inhibitor Ni2+ was used to investigate whether the decrease in intracellular Ca2+ content with LY- 294002 could be due to inhibition of the NCX reverse- mode activity. The minimal effect of LY- 294002 with Ni2+ suggests that the primary effect of LY- 294002 on EC coupling occurs through inhibition of PI3K- mediated L- type Ca2+ channel activity.