The role of P21-activated kinase (Pak1) in sinus node function

Highlights

• The loss of p21-activated kinase (Pak1) results in sinoatrial node (SAN) bradycardia.

• Pak1 regulates the SAN pacemaker's membrane clock component by attenuating class II HDAC4 activity.

• During atrial fibrillation (AF), downregulation of Pak1 can contribute to SAN bradycardia through ERK1/2 activation.

Abstract

Sinoatrial node (SAN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. In the atrial muscle attenuated activity of p21-activated kinase 1 (Pak1) increases the risk for AF by enhancing NADPH oxidase 2 dependent production of reactive oxygen species (ROS). However, the role of Pak1 dependent ROS regulation in SAN function has not yet been determined. We hypothesize that Pak1 activity maintains SAN activity by regulating the expression of the hyperpolarization activated cyclic nucleotide gated cation channel (HCN).

To determine Pak1 dependent changes in heart rate (HR) regulation we quantified the intrinsic sinus rhythm in wild type (WT) and Pak1 deficient (Pak1^−/−) mice of both sexes in vivo and in isolated Langendorff perfused hearts. Pak1^−/− hearts displayed an attenuated HR in vivo after autonomic blockage and in isolated hearts. The contribution of the Ca²⁺ clock to pacemaker activity remained unchanged, but Ivabradine (3 μM), a blocker of HCN channels that are a membrane clock component, eliminated the differences in SAN activity between WT and Pak1^−/− hearts. Reduced HCN4 expression was confirmed in Pak1^−/− right atria. The reduced HCN activity in Pak1^−/− could be rescued by class II HDAC inhibition (LMK235), ROS scavenging (TEMPOL) or attenuation of Extracellular Signal-Regulated Kinase (ERK) 1/2 activity (SCH772984). No sex specific differences in Pak1 dependent SAN regulation were determined.

Our results establish Pak1 as a class II HDAC regulator and a potential therapeutic target to attenuate SAN bradycardia and AF susceptibility.

Introduction

The heart's pacemaker, the sinoatrial node (SAN), consists of specialized muscle fibers that rhythmically generate electrical activity. This basal automaticity or intrinsic sinus rhythm (SR) is under constant control of the autonomic nervous system (ANS). SAN bradycardia, a manifestation of sinus node dysfunction (SND), describes the inability of the heart's natural pacemaker to generate cardiac excitation at an appropriate frequency [1]. SND and atrial arrhythmia frequently coincide [[1], [2], [3]] and SND itself increases the hazard ratio for atrial fibrillation (AF) by promoting dispersion of repolarization, reentry, and atrial ectopy [[4], [5], [6]]. In patients with AF and animal models of atrial tachyarrhythmia on the other hand, SAN automaticity is attenuated but can recover after cardioversion indicating an arrhythmia induced remodeling of the pacemaker mechanism [5,7,8].

The SAN pacemaker cells exhibit distinct electrophysiological and calcium (Ca²⁺) handling properties that allow them to rhythmically generate action potentials (APs). Due to the scarcity of the inward rectifier potassium (K⁺) channel (I_K1, Kir2.1–2.4), SAN cells exhibit a depolarized maximum diastolic potential and a progressive depolarization of the resting membrane potential (V_m) during diastole (diastolic depolarization). The diastolic depolarization is driven by two coupled cellular pacemaker mechanisms that have been termed the Ca²⁺- and membrane- clock [1,9,10]. Due to their coordinated action and interdependence, they make up the coupled-clock mechanism.

The contribution of the Ca²⁺ clock to SAN pacemaker activity depends on the release of Ca²⁺ from the sarcoplasmic reticulum through the activation of ryanodine or inositol 1,4,5-tris phosphate receptor (RyR and IP₃R, respectively) channels [[9], [10], [11], [12]]. These release events that increase in frequency toward the end of diastole [12,13], are translated into a depolarization of V_m by the electrogenic sodium‑calcium exchanger (NCX) which in the forward mode extrudes one Ca²⁺ ion by bringing 3 Na⁺ ions into the cell [11,[13], [14], [15]]. The rate of depolarization by the Ca²⁺ clock depends on the interplay between the Ca²⁺-load of the SR, the open probability of the Ca²⁺ release channels, as well as the expression and activity of NCX [10,16]. One driver of the membrane clock is the pacemaker current (I_f) through the hyperpolarization-activated, cyclic-nucleotide gated cation channel (HCN) [4,[17], [18], [19]]. The current is activated upon repolarization, counters further hyperpolarization of V_m, and contributes to the early phase of the diastolic depolarization [4,17]. Of the 4 HCN protein isoforms identified, HCN4 predominates in the human and rodent SAN and its deletion in animal models leads to a significant reduction of I_f, SAN bradycardia, and frequent sinus pauses [18,20,21]. In humans, loss of function mutations in the HCN4 channel or its auxiliary proteins have been associated with SAN bradycardia as well as AF, AV-block, and tachy-bradycardia [20,22,23]. In addition, voltage-dependent Ca²⁺ channels (VDCCs), tetrodotoxin (TTX)-sensitive Na⁺ channels (I_Na,TTX) [24,25], as well as transient receptor potential (TRPM and TRPC) [26] channels have been shown to contribute to the membrane clock. The T- type (Ca_v3.1) and L-type (Ca_v1.2, Ca_v1.3) channels expressed in SAN tissue, not only contribute to the membrane clock by furthering the depolarization, but also critically modulate the Ca clock mechanism. VDCC dependent Ca influx regulates the SR load and thereby the RyR open probability, and the frequency of the spontaneous Ca release events [13,[27], [28], [29]].

The serine/threonine protein kinase p21-activated kinase (Pak1), activated by Ras related small G-protein, exerts cardioprotective signaling [[30], [31], [32], [33], [34], [35]]. Pak1 attenuates cardiac hypertrophic remodeling by counteracting mitogen-activated protein kinase (MAPK) activity [34] and in atrial and ventricular myocytes we demonstrated that Pak1 regulates the production of reactive oxygen species (ROS) by antagonizing the activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2). Attenuation of Pak1 activity consequently increases cellular ROS production and the propensity for arrhythmic Ca²⁺ release in the atria and ventricle after ischemia reperfusion injury [33,35].

A ROS dependent remodeling of SAN pacemaker activity has been described in animal models of hypertension [36,37], diabetes [38], ischemia reperfusion [39], and cardiomyopathy [40]. The disease induced SAN bradycardia was shown to be a consequence of enhanced NOX2 as well as mitochondrial ROS production [36,39,40]. The ROS mediated reduction in pacemaker function was attributed to altered activity of the Ca²⁺ calmodulin kinase II (CaMKII) [37,41] and a subsequent increase in pacemaker cell apoptosis or a reduction in RyR open probability and subsequent attenuation of Ca²⁺ clock activity, respectively. As an alternative mechanism, a ROS dependent increase in class II histone deacetylase 4 (HDAC4) activity was linked to attenuated HCN4 protein expression [40]. Pak1 can regulate cardiac pacemaker activity by counteracting the positive chronotropic effect of β-adrenergic stimulation through activation of its downstream target protein phosphatase 2A (PP2A) and the reduction of L-type Ca²⁺ channel and inwardly rectifying K⁺-channel activity [42]. Under physiological conditions however, when the heart rate (HR) is under the influence of the ANS, no differences in pacemaker activity were determined between WT and Pak1^−/− animals [34,35]. The impact of Pak1 on SAN function in the absence of ANS control has yet to be determined. Since loss of Pak1 activity increases the susceptibility for AF and SAN dysfunction can be the cause and consequence of AF, in the current study, we aimed to determine the role of Pak1 in the regulation of cardiac pacemaker activity. In vivo, in the whole heart, and on the cellular level we tested the hypothesis, that Pak1 maintains SAN activity by regulating the expression of HCN through attenuation of NOX2 dependent ROS production and suppression of class II HDAC activity.