The Molecular Mechanism Of K2P1 Two Pore-domain Potassium Channel Inducing Fatal Arrhythmias In Hypokalaemia

Objective To explore the arrhythmias induced by K2P1 two pore-domain potassium channel and the related molecular mechanisms in hypokalaemia.MethodsFirstly,Cre-loxp genetic engineering technology was used to build cardiacspecific K2P1-overexpressed mice.In vivo animal fluorescence imaging,semiquantitative real-time polymerase chain reaction(RT-PCR),Western blot and immunofluorescence experiments were used to validate whether the cardiac-specific K2P1-overexpressed mice had built successfully.Adult K2P1-Tg+/+ mice(8-14 weeks)(K2P1+/+: Cre mice induced by tamoxifen)were enrolled as an experimental group in the study,while the control group was wild-type(WT)and K2P1-Tg-/-(K2P1+/+: Cre mice without inducing by tamoxifen)matched with the same age and sex.Secondly,we raised mice a K+-deficient diet for 6 weeks.Under normal(before feeding,serum [K+]=4.1-6.1m M),moderate(serum [K+]=2.5-3.0m M)and severe hypokalemia(serum [K+]=2.0-2.5m M),telemetry electrocardiogram(ECG)was used to monitor the longterm ECG(12h)in a state of free movement,and the lead II ECG was used to record the short-time resting ECG(10min)of mice under isoflurane anesthesia.Moreover,the Mapping Lab electrical mapping system was applied to electrical mapping of the whole heart and the ventricular tissue to further explore the role of K2P1 in acute hypokalemia.Finally,to explore the mechanism of K2P1 in causing fatal arrhythmias,we acutely isolated ventricular myocytes from adult mice by the Langendorff perfusion system,and recorded resting membrane potentials and currents of the whole mouse cardiomyocytes under normal and hypokalemic conditions by patch-clamp.Results1.K2P1 cardiac-overexpressed model(K2P1-Tg +/+ mice)was successfully built.2.Compare to WT or K2P1-Tg-/-mice,higher proportion of K2P1-Tg+/+ mice recorded ventricular ectopty(VE).3.K2P1-Tg+/+ mice revealed a higher mortality during 6 weeks of chronic hypokalemia modeling,accompanied by higher frequency of polymorphic ventricular tachycardia(PVT).4.Compare to WT or K2P1-Tg-/-mice,K2P1-Tg chronic hypokalemic mice revealed a delayed conduction in atria,ventricle and atrioventricular junction,and showed a prolonged P,QT,and PR interval.5.In the perfusion process of 2 m M [K+]e KH solution for 30 minutes,hypokalemiainduced sustained ventricular fibrillation(SVF)occurred earlier in K2P1-Tg+/+ hearts,and various types of VE happened more frequently.Activation maps of K2P1-Tg+/+ mice suggested that the pacing position deviated relative to sinus rhythm since VE happened,and multiple ectopic pacemakers were presented in a mouse during hypokalemia.In addition,spontaneous pacing activities(SEPs)were recorded in a higher proportion of the isolated K2P1-Tg +/+ ventricular tissues under hypokalemia.6.In acute isolated ventricular cardiomyocytes,K2P1-Tg+/+ group recorded a higher proportion of spontaneous action potentials(SAPs),and inward Na+ currents(blocked by qunine)in the 0 m M [K+]e extracellular solution(2m M Co Cl2 and 5μM TTX had been added),while no similar inward currents were recorded in the other two groups.Conclusion1.K2P1 mediates inward sodium currents under hypokalemia,which induces SAPs of ventricular myocytes,and finally leads to VE and fatal arrhythmia.2.K2P1 leads to a delayed conduction in ventricle and atrioventricular junction under hypokalemia,which may indirectly induce VE3.The mechanism of K2P1 leading to fatal arrhythmia in acute hypokalemia is essentially consistent with that in chronic hypokalemia.