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J Mol Cell Cardiol. 2014 Feb;67:77-85. doi: 10.1016/j.yjmcc.2013.12.017. Epub 2013 Dec 28.

p21-Activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes.

Author information

1
Center for Cardiovascular Research, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USA; Department of Medicine, Section of Cardiology, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USA.
2
Center for Cardiovascular Research, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USA; Department of Medicine, Section of Cardiology, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USA; Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USA.
3
Center for Cardiovascular Research, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USA; Department of Physiology and Biophysics, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USA.
4
Center for Cardiovascular Research, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USA; Department of Medicine, Section of Cardiology, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USA. Electronic address: kbanach@uic.edu.

Abstract

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 Ca(2+) 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 Ca(2+) 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 [Ca(2+)]i, which resulted in spontaneous Ca(2+) release events and waves. The Ca(2+) 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; p47(phox-/-)) 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 Ca(2+) 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 Ca(2+) overload under conditions where no significant changes in excitation-contraction coupling are yet evident.

KEYWORDS:

Arrhythmia; Cardiomyocyte; Excitation–contraction coupling; Ischemia; Sodium calcium exchanger; p21-Activated kinase 1

PMID:
24380729
PMCID:
PMC3930036
DOI:
10.1016/j.yjmcc.2013.12.017
[Indexed for MEDLINE]
Free PMC Article

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