P21-Activated Kinase (Pak1) is a Direct Modulator of Cardiac Excitation-Contraction Coupling (ECC) Gain

Ras related proteins regulate the activity of the serine-threonine protein kinase Pak1, which has been implicated in the regulation of cytoskeletal dynamics and motility. Recent evidence points to its role in cardiac ECC by attenuation of the β-adrenergic stimulation of ICa,L and IKr through activation of the phosphatase PP2A. To further determine the role of Pak1 in cardiac ECC we analyzed the Ca handling properties of isolated ventricular myocytes (VM) from Pak1-/- mice by laser scanning confocal microscopy. Isolated VMs from Pak1-/- mice exhibited a reduced Ca transient amplitude (Δ F/F0 WT: 1.78 ± 0.22 n=10; Pak1-/-: 1.26 ± 0.18 n=7, p<0.05) that was not based on a decrease in the load of the sarcoplasmic reticulum (WT: 5.58 ± 0.32; Pak1-/-: 4.82 ± 0.31) or a decrease in the current density of ICa,L (@-10mV: WT: 4.1± 0.5 n=11 pA/pF; Pak1-/-: 4.02± 1.6 n=9). However, the rise time of the Ca transient in Pak1-/- myocytes was significantly delayed (WT: 79 ± 5 ms; Pak1-/-: 119 ± 7 ms, p<0.05). The reduced amplitude could be based on a modified gain between Ca influx and Ca induced Ca release through the ryanodine receptor. β-adrenergic stimulation with isoproterenol (100 nM) not only rescued the Ca transient rise time in Pak1-/- myocytes but induced an exaggerated increase in Ca-transient amplitude (F/F0: WT: 4.45±0.25; Pak1-/-: 5.22±0.26; p<0.05) and decrease in tau (WT: 159.8±8ms; Pak1-/-: 121±3ms; p<0.05). The direct involvement of Pak1 in this process is suggested by the reversal of all parameters to control conditions by adenoviral overexpression of Pak1 in Pak1-/- VMs. Our results further support the role of Pak1 as a modulator of β-adrenergic stimulation in VMs and indicate a novel role in the maintenance of cardiac ECC gain.