| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2190575 | Journal of Molecular and Cellular Cardiology | 2014 | 9 Pages |
•Loss 1 of Pak1 activity increases basal ROS levels.•Pak1 is a negative regulator of NOX2 in cardiomyocytes.•NOX2 derived ROS increases NCX activity.•Latent increase in NCX activity predisposes cardiomyocytes to Ca2+ overload.
Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca2+ handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1−/−) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia.The Ca2+ transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1−/− VMs during 15 min of simulated ischemia. However, Pak1−/− VMs exhibited an exaggerated increase in [Ca2+]i, which resulted in spontaneous Ca2+ release events and waves. The Ca2+ overload in Pak1−/− VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1−/− VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47phox −/−) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1−/− VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca2+ overload in Pak1−/− VMs could be mimicked by low concentrations of ouabain.Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca2+ overload under conditions where no significant changes in excitation–contraction coupling are yet evident.
