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

Abstract

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.