Abstract P1087: P21-activated Kinase 1 Regulates Intrinsic Pacemaker Activity Through HCN4 Expression

Introduction: Cardiac pacemaker activity generated in the sinoatrial node (SAN) depends on a coupled clock mechanism that consists of a calcium (Ca-) and a voltage clock (V m -clock). Attenuation of either clock results in bradycardia which increases the risk for atrial fibrillation (AF). We have previously demonstrated that p21-activated kinase 1 (Pak1) is a negative regulator of NADPH oxidase 2 dependent production of reactive oxygen species (ROS). Here we hypothesized that attenuated Pak1 activity increases the susceptibility for AF by attenuating SAN function. Methods: In wild type (WT) and Pak1 knock-out (Pak1 -/- ) mice (FVBN), ECGs and atrial electrograms were recorded in vivo and in isolated Langendorff (LD)-perfused hearts. Results: Pak1 -/- compared to WT mice exhibited an attenuated intrinsic HR in vivo after autonomic blockage with atropine (1 mg/kg) and propranolol (1 mg/kg) (WT: 399 ± 8.05 bpm, n=16; Pak1 -/- : 365 ± 9.6 bpm, n=13, p<0.05) and in the isolated heart (WT: 343 ± 8.5 bpm, n=24; Pak1 -/- : 280 ± 6.03 bpm, n=28, p<0.01), suggesting attenuated SAN impulse generation. Block of the Ca-clock (cyclopiazonic acid (CPA): 5 μM) did not eliminate the difference in HR. Block of the V m -clock with the hyperpolarization activated cyclic nucleotide gated channel (HCN4) blocker ivabradine (IVA: 3 μM) however, had an attenuated effect on HR in Pak1 -/- (WT IVA : -45 ± 2.5 %, n=6; Pak1 -/- IVA : -22 ± 3.2 %, n=7, p<0.001) and eliminated the difference in SAN frequency (WT IVA : 186 ± 17 bpm, n=6; Pak1 -/- IVA : 234 ± 15 bpm, n=7) supporting a decreased V m -clock function. Attenuated HCN4 expression in Pak1 -/- could be recovered by suppression of Histone deacetylases class II activity (LMK235: 5 mg/kg/day, 3 days); WT LMK : 322 ± 9.4 bpm, n = 5; Pak1 -/- LMK 314 ± 3.4 bpm, n= 5;) as well as by scavenging of ROS (TEMPOL supplemented drinking water, 2 mmol/L, 3 days; WT Tem : 332 ± 12.7 bpm, n = 5; Pak1 -/- Tem : 318 ± 22 bpm, n= 5). Both treatments, restored the contribution of V m -clock to pacemaker activity in Pak1 -/- animals (WT LMK : -38 ± 8.3%, n = 5; Pak1 -/- LMK : -38 ± 1.2%, n= 4; WT Tem : -46 ± 3.7 %, n = 5; Pak1 -/- Tem : -41 ± 2.5 %, n= 5) and attenuated AF inducibility. Conclusion: In both sexes attenuated Pak1 signaling results in SAN bradycardia by ROS/HDAC dependent suppression of HCN4 expression.