Chronic Regulation Mechanism Of Herg Potassium Channel By Angiotensin II

Cardiac hypertrophy, heart failure(HF) is a common complication inmultiple cardiovascular diseases including coronary heart disease, valvularheart disease, hypertension. The electrical instability increase in cardiachypertrophy, HF due to the pathological remodeling and thus is oftenaccompanied by a variety of arrhythmias. More than half of patients with HFdie suddenly because of the occurrence of malignant arrhythmias.The rennin-angiotensin system (RAS) plays a key role in maintainingcardiovascular homeostasis. Studies have proved evidence that RAScontributes to cardiac arrhythmias in a variety of cardiovascular diseases,including HF and cardiac hypertrophy. Angiotensin II (AngII) is the keycomponent of RAS, which can activate multiple intracellular signalingpathways mainly through the activation of the AT1receptor to regulatecardiovascular function. Experimental studies have demonstrated that rats withoverexpression of AngII gene die suddenly because of severe ventriculartachycardia. In addition, AngII down-regulates Itocurrent density in acutelyisolated canine and rat ventricular myocytes, as well as mammalian cellsexpressed with Kv4.3channel gene. These findings suggest AngII directlymodulates the function of ion channels by stimulating the AT1receptor, andinvolve in the pathological electrical remodeling.The delayed rectifier potassium current plays a key role in ventricularmyocytes action potential repolarization in mammal species. Thepore-forming subunit of the rapidly activating component (IKr) is encoded bythe human 'ether-a-go-go' related gene (hERG). The hERG potassiumchannel has an important influence on action potential shape and durationbecause of its special characteristics of the dynamics. The dysfunction of thechannel in disease status leads to the prolongation of action potential duration and/or deformation, which predispose the heart to ventricular tachyarrhythmia.Our previous studies have shown that AngII produces an inhibitory effecton IKr/hERG currents via AT1receptors linked to the PKC pathway in guineapig ventricular myocytes, suggesting elevated levels of AngII are involved inthe occurrence of arrhythmias in cadiac hypertrophy and HF. It is known thatvarious actions of AngII via AT1receptor can be divided into the short-termeffects that occur within minutes and the long-term effects that take placewithin hours or even later. We found that the abundance of the hERGpotassium channel mature protein reduced after long-term incubation of AngII(>12h)(unpublished data). However, little is known about the mechanismunderlying the down-regulation of AngII on the protein abundance.Rencent studies have proved that chronic stimulation of Gprotein-coupled receptors has significant effect on post-transcriptionregulation of hERG potassium. Therefore, the present study was designed toinvestigate post-transcription regulation (including protein forward traffickingand membrane protein degradation into the cell process) of AngII on hERGpotassium channel and analyse its underlying signal transduction mechanismin heterologous expression systems using molecular biological methods.Part1Post-transcriptional regulation of hERG potassium channel byangiotensin IIObjective: To investigate post-transcriptional regulation of hERGpotassium channel by AngII.Methods:(1) lipofectamine2000transient transfection: Stablytransfected hERG-HEK293cells were transiently transfected with AT1receptor Cdna plasmids using Lipofectamine2000and studied within24-48hof transfection.(2) Immunofluorescence: Cells were fixed with4%paraformaldehyde,then permeabilized with0.2%Triton X-100or skipped this step. After5%BSAblocked, incubated with the appropriate antibody (anti-hERG) andfluorescently labeled secondary antibody, observed by confocal microscopy.(3) Western blot: The extraction of total cellular proteins were resolved by SDS-PAGE and transferred to NC membranes, blocked with5%nonfatmilk, then incubated with the appropriate antibody and fluorescently labeledsecondary antibodies, scanned using Odyssey Infrared Imaging System.(4) In-cell western: Cells were fixed with paraformaldehyde thenblocked with5%nonfat milk, incubated with the appropriate antibody andfluorescently labeled secondary antibodies, then scanned using OdysseyInfrared Imaging System.Results:(1) Western blot analysis showed the band appeared in65kDa,which was consistent with molecular weight of AT1receptor, suggesting thatAT1receptor was successfully expressed in hERG-HEK293cells.(2) The effect of hERG potassium channel protein abundance bylong-term incubation of AngII: In-cell western detection showed that themature protein in membrane was decreased after24hours incubation of AngII(100nM)(69%±4%,P<0.01). To investigate hERG protein in the cellmembrane and intracellular distribution, we used immunofluorescencestaining by confocal microscopy and found that AngII significantly reducedthe non-permeabilized or permeabilized cell membrane fluorescence. However,the cytoplasmic fluorescence of permeabilized cell did not changesignificantly.(3) The effect of hERG potassium channel protein forward trafficking byAngII: Western blot analysis showed that the mature protein wassignificantly reduced(68%±4%, P<0.01), but the immature protein did notchange significantly(P>0.05). It is known that Fluoxetine (3μM) or hERGpotassium channel mutants A422T and H562P cause trafficking disturbance.Our result was consistent with previous study. Western blot analysis showedthe mature protein was significantly reduced(64%±2%,P<0.01), with theimmature protein significantly increased(114%±5%,P<0.05). It suggestedAngII did not affect hERG potassium channel protein forward trafficking.(4) The effect of hERG potassium channel membrane proteindegradation by AngII: We blocked forward trafficking with the Golgi transitinhibitor brefield A (BFA,10μM) to observe whether AngII affect degradation process or not. The control group in2h,4h,8h,12h,24h decreased to82±8%,70%±1%,63%±9%,55%±8%,53%±8%(P<0.05),the AngII group in2h,4h,8h,12h,24h decreased to77%±7%,64%±9%,55%±11%,44%±12%,40%±12%(P<0.05), suggesting that AngII accelerated the degradation ofmembrane protein. Lactacystin (5μM, proteasome inhibitor) significantlyinhibited AngII-mediated reduction of the mature protein(67%±4%increasedto89%±3%,P<0.05), but Bafilomycin (1μM, lysosomal inhibitor) had nosignificant effect on the role of AngII(P>0.05). It suggested that AngIIaccelerated protein degradation, mainly through the proteasome pathway.Conclusions: In heterologous expression systems, the chronic incubationof AngII can significantly reduce the hERG potassium channel membranemature protein abundance. The reason may be caused by acceleratedmembrane protein degradation through the proteasome pathway.Part2Signal transduction mechanisms of angiotensin II mediated hERGpotassium channel membrane protein downregulationObjective：To analyze the signal transduction mechanism of angiotensinII mediated hERG potassium channel membrane protein downregulation.Methods: the same as the first part.Results: Westen blot showed:(1) PKC inhibitor Bis I (100nM)significantly inhibited AngII-mediated reduction of the mature protein(64%±4%increased to90%±4%, P<0.01)).(2) PKC activator PMA (100nM) and OAG(100nM) could alsosignificantly inhibited AngII-mediated reduction of the mature protein(59%±5%increased to101%±5%,P<0.01；58%±4%increased to100%±5%,P<0.05).(3) PKA inhibitor H-89(1μM) could not inhibit AngII-mediatedreduction of the mature protein(P>0.05).Conclusion: In heterologous expression systems, the chronic incubationof AngII reduce the hERG potassium channel membrane mature protein ismainly mediated by PKC signaling pathway.