Modulation And Underlying Signal Transduction Mechanisms Of Angiotensin â…¡on The Slow Component Of Delayed Retifier Potassium Current

Ventricular arrhythmias are common in both cardiac hypertrophy and failure. Cardiac failure in particular is associated with a significant increase in the risk of sudden cardiac death. It is known that cardiac myocytes either from hypertrophic or failing heart exhibit remarkable electrophysiological remodeling, which is characteristic of the prolongation of action potential. A reduction in the potassium currents has been the most consistent finding in a variety of experimental models and species with cardiac hypertrophy and failure. The renin–angiotensin system (RAS) plays an important role in the development of cardiac hypertrophy via the activity of its effector, angiotensin II (AngII). Although it is well recognized that Ang II exerts direct actions on cardiac tissues inducing cardiomyocyte hypertrophy and mechanical dysfunction, relatively little is known concerning the role of Ang II in the development of arrhythmias and cardiomyocyte electrical dysfunction.There is increasing evidence that the RAS is associated with the occurrence of atrial and ventricular arrhythmias in experimental animals. It has been shown that Ang II downregulates transient outward potassium current (Ito) density in isolated rat and canine ventricular myocytes or mammalian cells expressed with Kv4.3 channel gene. It has been demonstrated that specific overexpress of Ang II gene in mouse heart tissue can prolong action potential and increase the incidence of ventricular arrhythmias, without increase of the circulating levels of Ang II. The similar result has been reported by Rivard et al that mice with cardiac-specific overexpression of human Ang II type 1 (AT1) receptor show a significant decrease in the amplitude of Ito, ultra-activated delayed rectifier current (IKur) and inward rectifier current (IK1) with the prolongation of action potential duration (APD) and spontaneous ventricular arrhythmias. Particularly, the young transgenic animals display delayed repolarization and a high incidence of arrhythmias without cardiac hypertrophic remodelling. The results indicate that electrophysiological changes are not secondary to cardiac remodelling, but a direct response to the stimulation on AT1 receptor. These results suggest that Ang II directly modulates ion channels and thus contributes to the pathological electrical remodeling.The delayed rectifier K+ currents (IK) are the major repolarizing outward currents of ventricular action potentials in mammalian species, including humans and consist of rapidly and slowly activating components (IKr and IKs, respectively). It has been demonstrated that IKs is a heteromultimeric complex composed of four pore-formingαsubunits and two accessoryβsubunits encoded, respectively, by the KCNQ1 and KCNE1 genes (5, 7, 43). Dysfunction of IKs due to genetic mutations in the KCNQ1 or KCNE1 gene is linked to congenital long QT syndrome (LQT1 or LQT5). Electrical remodeling in diseased hearts is often associated with a reduction of IKs, e.g., myocardial infarction, chronic heart failure (29, 30) and cardiac hypertrophy (38, 55). All these conditions increase the risk for life-threatening ventricular arrhythmia. We previously reported Ang II produced inhibitory effect on IKr via AT1 receptor linked to PKC pathway in ventricular myocytes. However, available information is limited regarding the effect of Ang II on repolarizing IKs currents in cardiac ventricular myocytes. The present study was designed to examine the possible regulation of IKs by Ang II in isolated guinea pig ventricular myocytes and heterologous expression system using the whole-cell patch-clamp technique.Part 1 Inhibition of slow component of delayed rectifier potassium current in ventricular myocytes by angiotensin IIAim: To assess the effect of Ang II on IKs in guinea pig ventricular myocytes.Methods: Single ventricular myocytes were enzymatically dissociated from the heart of adult guinea pigs. The IKs was recorded by using the standard whole-cell patch-clamp technique. The Na+ current was inactivated by a holding potential of 4?0 mV, and the L-type Ca2+ current was blocked by 1μM Nimodipine (Nim). The external solution contained (in mM) NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.33, glucose 5.5 and HEPES 5 (pH adjusted to 7.4 with NaOH). The pipette solution contained (in mM) potassium aspartate 70, KCl 50, KH2PO4 10, MgSO4 1, Na2-ATP 3, Li2-GTP 0.1, EGTA 5, and HEPES 5 (pH adjusted to 7.2 with KOH). Ventricular myocytes were depolarized from a holding potential of -40 mV to various prepulse potentials of -30 to 60 mV for 2 s and repolarized to -40 mV to evoke outward tail currents in the presence of IKr blocker, E4031 (5μM). All experiments were performed at room temperature (22–23℃) using an Axopatch 200B amplifier. The electrical signals were sampled at 2.5-10 kHz and filtered at 1 KHz using a low-pass filter, and digitized with an A/D converter. A pClamp software (Version 8.1) was used to generate voltage-pulse protocols, acquire and analyze data.Results: AngII markedly reduced IKs during depolarizations and tail currents on return to the holding potential. The tail current densities at +40mV were 0.77±0.07 and 0.49±0.07pA/pF in the absence and presence of Ang II (100nM), respectively. The reduction of IKs tail current occurred within 2–3 min and reached saturation about 5-10 min after addition of Ang II (100nM) into the bath. AngII decreased the amplitude of IKs in a concentration-dependent manner with an IC50 of 8.29 nM. The voltage dependence of IKs activation was found to be not affected by AngII. The activation and deactivation time constants were not affected by AngII.Conclusion: AngII produced an acute inhibitory effect on IKs current in ventricular myocytes, which was not achieved through affecting the voltage-dependent activation, and kinetics of activation and deactivation of the channel.Part 2 AngII inhibites IKs via PKCεisoformAim: To further examine the signal transduction pathways mediating the inhibitory action of AngII on IKs.Methods: The IKs was recorded in guinea-pig ventricular myocytes by using the same patch-clamp technique as it mentioned in the first part.Results: Specific AT1 receptor blocker, losartan (1μM), antagonized the inhibitory action of Ang II on IKs. But, AT2 receptor blocker, PD123319 (1μM), had no any effect on the response of ventricular myocytes to Ang II. The result suggests that the inhibitory action of Ang II on IKs is mediated via AT1 receptor. It is known that AT1 receptors belong to G-protein coupled receptor family and are mainly coupled to heterotrimeric G protein q/11. Stimulation of AT1 receptors results in the activation of PKC and the increase in intracellular Ca2+. To investigate whether the actions of Ang II resulted from an increase of intracellular free Ca2+, ventricular myocytes were dialyzed via the patch pipette with an intracellular solution containing 20 mM BAPTA, a faster and more efficient Ca2+ chelator than EGTA . It was shown that Ang II still decreased the tail current amplitude of IKs, indicating that the intracellular Ca2+ release was not required for the inhibitory effect of Ang II on IKs. On the other hand, a non-selective inhibitor for PKC isoforms, stausporine and Bis-1, significantly attenuated the Ang II-induced decrease in the amplitude of IKs. In the presence of nonspecific PKC activators, PMA, Ang II produced little further effect on IKs. Specific PKA inhibitor, H-89, had no effect on the action of Ang II. In addition, The inhibition on Iks by Ang II was little affected by G?-6976 or G?-6983, furthermore, the inhibitory effect was significantly antagonized by an internal dialysis with PKCε-selective inhibitory peptideεV1-2. Therefore, the present study supports the view that activation of AT1 receptors is predominantly linked to PKCε-dependent pathway to mediate the inhibitory action of Ang II on IKs.Conclusion: Our results provided the direct experimental evidence that Ang II produced an acute inhibitory effect on IKs via the AT1 receptor in ventricular myocytes and PKCεwas the predominant isoform underlying the inhibition on the current.Part 3 Effects of Ang II on KCNQ1/KCNE1 currentAim: To further assess the effects of Ang II on IKs in heterologous expression system using the perforated patch-clamp technique. Methods: Perforated patch technique was used to record in HEK293.Results: AngII reduced KCNQ1/E1 during depolarizations and tail currents. The inhibition of Ang II on KCNQ1/E1 was abolished by AT1 receptor blocker, losartan (1μM). The voltage dependence of KCNQ1/E1 activation was found to be not affected by AngII.The response to Ang II was attenuated by inhibitor of PKC with Bis-1. AngII also reduced KCNQ1 currents. Bothαandβsubunits of KCNQ1/E1 may involve in the modulation of Ang II on the channel. KCNQ1/E1/E2 trimer kinesics property like KCNQ1/E1. The inhibitory effect of Ang II on KCNQ1/E1/E2 was significantly antagonized by KCNE2.Conclusion: Ang II inhibites KCNQ1/E1 current. The present study provides experimental evidence to suggest that, activation of PKC contributes to Ang II-mediated inhibition. Bothαandβsubunits of KCNQ1/E1 may involve in the modulation of Ang II on the channel.SUMMARY1. AngII produces an inhibitory effect on IKs and KCNQ1/KCNE1 currents via AT1 receptor linked to PKC pathway in ventricular myocytes and heterologous expression system,which is not a result from affecting the voltage-dependent activation, and kinetics of activation and deactivation of the channel.2. The PKCεisoform is the predominant isoform underlying the inhibitiory action of Ang II on IKs and KCNQ1/KCNE1 currents3. Bothαandβsubunits of KCNQ1/KCNE1 may involve in the modulation of Ang II on the channel.The result suggests a potential mechanism by which elevated levels of Ang II may be involved in the electrophysiological remodeling and thus occurrence of arrhythmias in the cardiac pathological conditions.