Specifically Enhancing Inward Rectifier Potassium Current (I_K1): A New Antiarrhythmic Pathway

Background: The inward rectifier K⁺ current (I_K1) plays a significant role in the final repolarization and resting phases of the ventricular action potential. The molecular basis of I_K1 has been ascribed to members of the Kir2.x sub-family of inward rectifier K⁺ channel genes. Near RMP, the ventricular I_K1 conductance is much larger than that of any other current except the ATP-sensitive potassium current(IKATP), which is normally not active. Dysfunction of the Kir2 channel or modulation of I_K1 would likely have profound effects on cardiac excitability and ventricular arrhythmogenesis. However, there have been no I_K1-specific pharmacologic agents so far.Objective: We would recognize zacopride as a specific I_K1 agonist in rat cardiomyocytes and detect the probable signaling pathway(s) involved in its action. Further, we attempted to justify whether enhancing, rather than blocking, I_K1 is a feasible pathway of antiarrhythmia.Methods and Results: By whole-cell patch-clamp technique in rat or guinea pig cardiomyocytes, zacopride at 0.1-10μmol/L enhanced I_K1 without effects on Ca+ current (ICa-L), Na⁺ current (INa), transient outward K⁺ current (Ito), sustained outward delayed rectifier K⁺ current (Isus), delayed rectifier K⁺ current (IK), Na⁺-Ca2+ exchanger (NCX) current (INa/Ca), Na⁺-K⁺ pump current (Ipump) and IKATP. Both inward and outward component of I_K1 were increased by zacopride in a concentration-relative and reversible manner (P<0.05). 1.0μmol/L Zacopride showed the maximal effect on I_K1 with the mean increment by 33.8% in inward current (at -100mV) and 32.4% in outward current (at -60mV), and the effect could be abolished by 1μmol/L BaCl2 (P<0.05). Correspondingly, zacopride at 1.0μmol/L hyperpolarized resting membrane potential (RMP) from -81.3±0.9 mV up to -87.5±1.7mV and shortened action potential duration (APD90) from 32.48±2.70 ms to 25.61±3.97 ms (n=6, P<0.01). The enhancement of I_K1 by zacopride could not be inhibited by 5-HT4-receptor antagonist RS23597-190 and 5-HT3-receptor agonist m-chlorophenylbiguanide at 10μmol/L respectively. Furthermore, PKA inhibitor KT5720 (5μmol/L) could inhibited the effect of zacopride on I_K1(P<0.05), while PKC inhibitor GF109203X and PKG inhibitor KT5823 at 5μmol/L respectively were short of effects on augmented I_K1 by zacopride (P>0.05). In rat papillary muscles, zacopride significantly decreased the incidence of delayed afterdepolarizations (DADs) and triggered activity (TA) induced by isoproterenol (Iso) from 85.7% to 28.6% (n=14, P<0.05). Langendorff-perfused hearts were subjected to either coronary artery occlusion for 15 minutes or 15 minutes followed by 15 minutes reperfusion to induce definite arrhythmia. Zacopride at different concentration (0.1-10μmol/L) was added at 3 minutes before ischemia or reperfusion manipulating to explore its possible effect on the experimental rhythm disturbances. During ischemia, the total of PVB, the duration of VT and VF, the incidence of VT and VF were concentration-relatively decreased by zacopride treatment. The largest antiarrhythmic effect appeared at the concentration of 1.0μmol/L which consisted with the most effective concentration of zacopride on I_K1. At 1.0μmol/L, zacopride reduced the incidence of ventricular tachycardia (VT) from 100% to 25% (P<0.01), the incidence of ventricular fibrillation from 75% to 12.5% (P<0.01), the duration of VT from 8.58±3.65s to 2.69±2.24s (P<0.01), the duration of VF from 23.51±15.77s to 1.67±1.33s (P<0.01), and the number of premature ventricular beats (PVB) from 173±26 to 9±4 (P<0.01). Based on patch clamp result that 1.0μmol/L BaCl2 could abolish the I_K1 increment induced by 1.0μmol/L zacopride, 1.0μmol/L BaCl2 co-applied with 1.0μmol/L zacopride reversed the antiarrhythmic effect of zacopride as well. In BaCl2-treatment group, the total of PVB (45±19), the duration of VT (7.21±4.67s) and VF (11.87±9.43s), the incidence of VT (50%) and VF (56.3%) were significantly increased compared with 1μmol/L zacopride-treated rats (P<0.01). Similar protection was observed during reperfusion with a most effective concentration of zacopride at 0.1μmol/L which was 10 times less than that on ischemic arrhythmia. In the presence of 0.1μmol/L zacopride, the incidence of VT declined from 100% to 31.3% (P<0.01), VF declined from 75% to 12.5%, and the duration of VT shortened from 21.45±4.85s to 2.31±1.87s, the duration of VF shortened from 76.4±4.91s to 0.88±3.57s, the number of PVB decreased from 107±27 to11±9 (n=16, P<0.01). The antiarrhythmic effects were partly abolished by 1μmol/L BaCl2 (P<0.01). The antiarrhythmic effects of zacopride were also observed in anesthetized rats underwent 15-minute occlusion or 15-minute followed by 15-minute reperfusion. Far more dramatic protection was observed during ischemia. At the dose of 15μg/kg, zacopride showed the most potent antiarrhythmic action that compared favourably with lidocaine (7.5mg/kg), a classical antiarrhythmic agent. With prophylactic usage of 15μg/kg zacopride, the onset of arrhythmia was delayed from controlled 344.13±59.79s to 475.63±99.06s after ligation of coronary artery, the number of PVB decreased from 149±65 to 45±42, the incidence of VT declined from 100% to 25%, and the duration of VT shortened from 51.18±45.61s to 2.09±3.91s (n=8, P<0.05). None rat exhibited VF while 75% rats in control group developed VF with the mean duration of 6.90±7.05s (P<0.05). Less protection was observed during reperfusion. Zacopride showed the largest protective effects at dose of 1.5μg/kg that was 10 times less than that on ischemic arrhythmias. The incidence of VT declined from controlled 100% to 32.5%, the incidence of VF declined from 87.5% to 12.5%, and the duration of VT shortened from 13.35±8.98s to 5.10±4.62s, the duration of VF shortened from 11.72±8.91s to 1.25±3.54s (n=8, P<0.05). There was no significant effect on the total of PVB (p>0.05). Zacopride also significantly shortened the duration of aconitine-elicited arrhythmias from 48.18±2.32 min to 20.12±1.29 min (n=8, P<0.01) in ex vivo hearts and from 56.83±3.46 min to 37.57±2.62 min (n=8, P<0.01) in in vivo rats.Conclusions:1. Zacopride is firstly recognized as a specific agonist of I_K1 in rat ventricular myocytes. At 0.1-10μmol/L, zacopride enhanced both inward and outward component of I_K1 in a concentration-relative manner with no significant effects on I_Ca-L, I_Na, I_to, I_sus, I_Na/Ca, I_pump, I_KATP in rat and IK in guinea pig.2. The augmentation of I_K1 by zacopride might be via a PKA-mediated signaling pathway which is independent on 5-HT4 and 5-HT3 receptors.3. Zacopride at 0.1-10μmol/L could significantly hyperpolarize the resting membrane potential, increase the action potential amplitude and shorten the action potential duration.4. Zacopride at 1μmol/L could significantly decrease the incidence of Iso-induced delayed afterdepolari zations (DADs) and triggered activity (TA) .5. Zacopride could remarkably suppress various ventricular arrhythmias induced by ischemia, reperfusion or aconitine.6. Zacopride, as a specific agonist of I_K1, is not only a potential antiarrhythmic agent but also a valuable pharmacological tool in the experimental study.