Regulation of L-Type Calcium Channel and Delayed Rectifier Potassium Channel Activity by p ²¹ -Activated Kinase-1 in Guinea Pig Sinoatrial Node Pacemaker Cells

Abstract

Abstract-Phosphorylation of ion channels plays an important role in the regulation of cardiac function, but signaling mechanisms controlling dephosphorylation are not well understood. We have tested the hypothesis that p 21 -activated kinase-1 (Pak1), a serine-threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity through a mechanism involving protein phosphatase 2A. We report a novel role of Pak1-mediated signaling in attenuating isoproterenol-induced enhancement of L-type Ca 2ϩ current (I CaL ) and delayed rectifier potassium current (I K ) in guinea pig SAN pacemaker cells. We demonstrate that in guinea pig SAN: (1) ignaling pathways activated by ␤-adrenoceptors that increase heart rate have been well studied (reviewed elsewhere 1-4 ), but parasympathetic pathways that attenuate this signaling and cause a slowing of heart rate are less well understood. Although there is evidence that the antiadrenergic effect of acetylcholine is achieved at least in part by a depression in adenylate cyclase activity and reduced cAMP levels mediated via inhibitory G proteins, 5 emerging evidence suggests an important role for a signaling pathway involving protein phosphatase 2A (PP2A). 6,7 This idea fits with evidence indicating a dynamic balance between kinase and phosphatase activity in the control of cardiac ion channel phosphorylation and dephosphorylation even in the absence of autonomic stimulation. 8 -11 Pathways regulating the phosphorylation of ion channels in cardiac cells are relatively well understood, 12 but possible signaling pathways controlling dephosphorylation remain unclear.We have tested the hypothesis that signaling through p 21 -activated kinase-1 (Pak1), a serine-threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity. Our hypothesis is based on our demonstration that Pak1 binds to PP2A and induces dephosphorylation of cardiac myofilament proteins. 7 Results of our approach, which involved infection of SAN pacemaker cells with an adenovirus expressing constitutively active Pak1 (Ad-Pak1), support this hypothesis. Materials and Methods AnimalsAge-matched (12-week-old) female guinea pigs were purchased from Charles River (Wilmington, Mass; Margate Kent, UK); procedures were performed in accordance with national and institutional guidelines (US and UK). Viral InfectionFreshly isolated guinea pig SAN pacemaker cells were prepared by procedures based on previous methods 13 and were allowed to settle onto laminin-coated coverslips in 6-well tissue culture plates. These cells were cultured for 2 hours in RPMI medium 1640ϩ4% FBS Original

Small G Proteins in the Cardiovascular System: Physiological and Pathological Aspects

“…Similarly, in the resting condition, the outward K ϩ current was not changed in SAN cells expressing active Pak1, but the ␤-adrenergic stimulation-induced increase in K ϩ current amplitude and acceleration of the K ϩ current decay was almost abolished. In whole heart preparations, overexpression of active Pak1 attenuated the positive chronotropic action of ␤-adrenergic stimulation (219).…”

“…Endogenous Pak1 protein is abundant in SAN, atrial, and ventricular tissue (219). Expression of active Pak1 does not modify the amplitude of the L-type Ca 2ϩ current in SAN cells but strongly reduces the stimulatory effect of ␤-adrenergic stimulation (219).…”

“…Endogenous Pak1 protein is abundant in SAN, atrial, and ventricular tissue (219). Expression of active Pak1 does not modify the amplitude of the L-type Ca 2ϩ current in SAN cells but strongly reduces the stimulatory effect of ␤-adrenergic stimulation (219). Similarly, in the resting condition, the outward K ϩ current was not changed in SAN cells expressing active Pak1, but the ␤-adrenergic stimulation-induced increase in K ϩ current amplitude and acceleration of the K ϩ current decay was almost abolished.…”

Cardiomyocyte-specific overexpression of an active form of Rac predisposes the heart to increased myocardial stunning and ischemia-reperfusion injury

“…2 and Table 1), i.e., these mice showed increased susceptibility to I/R injury. Here, it is noteworthy that PAK1 activity is increased by hypoxia/reoxygenation in cultured cardiomyocytes (43) and PAK1 can blunt ␤-adrenergic pacemaker activity in myocardial cells (30). Increased susceptibility with stunning was consistently observed in a clinically relevant model of acute in vivo I/R where MI was significantly larger in TG mice (Fig.…”

p21-activated kinase improves cardiac contractility during ischemia-reperfusion concomitant with changes in troponin-T and myosin light chain 2 phosphorylation

“…Pak1 activates protein phosphatase 2a (PP2A) (11,30) and leads to the dephosphorylation of cardiac proteins, including cardiac troponin-I (cTnI) and myosin-binding protein C (MyBP-C) (11). The dephosphorylation of cTnI and MyBP-C increases myofilament Ca 2ϩ sensitivity in adult rat cardiac myocytes (10,11) and in cardiac intact trabeculae (29). Activation of Pak1 is not associated with a change in phospholamban phosphorylation (25).…”