title: p21-Activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes.
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.
authors.affiliation: 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.
authors.forename: Jaime
authors.initials: J
authors.lastname: DeSantiago
authors.affiliation: 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.
authors.forename: Dan J
authors.initials: DJ
authors.lastname: Bare
authors.affiliation: 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.
authors.forename: Lei
authors.initials: L
authors.lastname: Xiao
authors.affiliation: 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.
authors.forename: Yunbo
authors.initials: Y
authors.lastname: Ke
authors.affiliation: 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.
authors.forename: R John
authors.initials: RJ
authors.lastname: Solaro
authors.affiliation: 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.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Banach
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006352:Heart Ventricles / Q000201:enzymology; D015151:Immunoblotting; D051379:Mice; D015428:Myocardial Reperfusion Injury / Q000503:physiopathology; D032383:Myocytes, Cardiac / Q000201:enzymology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D019255:NADPH Oxidases; D054462:p21-Activated Kinases
keywords: Arrhythmia; Cardiomyocyte; Excitation–contraction coupling; Ischemia; Sodium calcium exchanger; p21-Activated kinase 1