The Effect Of Angiotensin ? On The Small Conductance Calcium-activated Potassium Channel In Rat Cardiomyocytes

Objective: Activation of the renin-angiotension-aldosterone(RAAS)System has associated with atrial fibrillation occurrence,which induce atrial electrical remodeling and structural remodeling.Small conductance calcium-activated potassium channels(SK channels)are closely related with development and progression of atrial fibrillation(AF),SK2 is the main subunits of SK channels.Studies have shown that the SK channel blocker NS8593 significantly reduces the incidence and the duration of AF in atrial rapid-pacing canine models.The previous results of our group also showed the relation between activation of RAAS and AF occurrence.Chronic treatment with angiotensin ?(Ang ?)induced increase of Ang ? concentration both in serum and left atrial tissues in rapid pacing canine model.Ang? also significantly increased AF duration and frequencies of AF occurrence during rapid pacing.while,pacing + Ang? + Valsartan group can significantly inhibited this effect.SK channel blocker NS8593 significantly inhibited the frequences and duration of AF in the pacing + Ang? group.Therefore,Ang? may participate in the development of AF through the remodeling of the SK2 channel,The molecular mechanism of Ang? on SK2 channel is still unclear.Methods:(1)qRT-PCR and western blotting techniques were used to detect the gene expression levels and the protein expression levels of Ang? on SK2 channel in rat atrial tissues and myocytes.(1)The Ang? was treated with mini-osmotic pump(200ng/kg/min)for 14 days.Atrial tissue was removed after treatment with Ang? for 14 days in a chronic treatment rat model.qRT-PCR and western blotting were used to detected the gene and protein expressions of SK2 channels.(2)The atrial myocytes from neonatal rats were isolated by combination with trypsin and type ? collagenase.Atrial myocytes were randomly divided into control group,1.5?M Ang? group,1.5?M Ang? + 10?M Valsartan group,1.5?M Ang? + 10?M PD123 319 group.After treatment for 72 h,the gene and protein expression of SK2 were detected by qRT-PCR and Western blotting techniques,respectively.(2)The effect of Ang? on SK2 channel membrane protein expression changes were examined by Biotin-Avidin-System in the rat atrial myocytes and in transient transfected KCNN2 gene HEK293 T cell line.(3)The interaction between SK2 channel protein and AT1 R or AT2 R was detected by immunoprecipitation in co-transfected SK2 and AR1 R or AT2 R in HEK293 T cell line.(4)The protein traficking of SK2 from rat atrial myocytes were detected by immunofluorescence and total internal reflection imaging.(5)Flow cytometry technique was also used to detect the trafficking of SK2 channel protein on HEK293 T cell line.(6)The effects of Ang? on SK2 current were detected by whole-cell patch clamp technique with co-transfected SK2 and AR1 R or AT2 R in HEK293 T cell line.Results:(1)Ang? increased the gene and protein expressions of of SK2 channel in rat atrial tissues and atrial myocytes:(1)Ang? increased the gene expression level of KCNN2(n=7,P<0.05)and the total protein expression level of SK2 channel in atrial tissues from SD(n=7,P<0.01);(2)Ang?(1.5?M)increased KCNN2 gene expression in neonatal rat atrial tissue(n=5,P<0.01),and 10 ?M Valsartan reversed this effect(n=5,P<0.01);Similarly,1.5?M Ang? also up-regulated the expression level of SK2 channel protein in neonatal atrial myocytes(n=5,P<0.01),which was also reversed by 10?M Valsartan(n=5,P<0.01);(2)Ang? increased the SK2 channel membrane protein expression level:(1)1.5?M Ang ? increased the SK2 channel membrane protein expression in neonatal atrial myocytes(n=6,P<0.01),and was reversed by 10 ?M Valsartan(n=5,P<0.05).(2)Co-transfected with SK2 and AT1 R in HEK 293 T cell line,1.5?M Ang? increased the membrane protein expression of SK2 channel(n=5,P<0.05);while co-transfected with SK2 and AT2 R in HEK 293 T cell line,Ang?(1.5?M)was not increased membrane protein in SK2 channels(n=4,P>0.05).(3)Protein-protein interactions between SK2 and AT1R: SK2 interactions with AT1R/AT2 R were detected by co-immunoprecipitation with the HEK293 T cell line co-transfected with SK2 and AT1 R or co-transfected with SK2 and AT2 R.There is a protein-protein interaction between SK2 and AT1 R.while,there is no significant interaction between SK2 and AT2 R.(4)Ang?(1.5?M)promoted the SK2 channels trafficking from intracellular to plasma membrane:(1)Ang ?(1.5?M)promoted the SK2 channel trafficking to plasma membrane in neonatal rat atrial myocytes(n=10,P<0.01);(2)Co-transfected with SK2 and AT1 R,Ang?(1.5?M)promoted the SK2 trafficking from intracellular to the plasma membrane(n=3,P<0.01);However,HEK293 T cells co-transfected with SK2 and AT2 R,Ang?(1.5?M)had no significant effect on the SK2 trafficking(n=3,P>0.05);In HEK293 T cells,transfected with KCNN2 alone,Ang?(1.5?M)also had no significant effect on the trafficking of SK2(n=3,P>0.05).(5)The effect of Ang? on current amplitude of SK2 channel:(1)Ang?(1.5?M)increased SK2 channel current amplitude on CHOK1-SK2-GFP cell line transfected with AT1R;(2)There was no significant effect of Ang ? on the SK2 channel current amplitude in the cell line transfected with AT2 R CHOK1-SK2-GFP;(3)1.5 ?M Ang? also had no significant effect on CHOK1-SK2-GFP alone,without transfected AT1 or AT2 receptor.Conclusion: RAAS activation is closely related to the occurrence of atrial fibrillation.Ang? targeted AT1 R promotes the upregulation of SK2 channel protein by trafficking from intracellular to the plasma membrane.The effected of Ang? on SK2 channel is involved in atrial electrical remodeling,which maybe one of the mechanism of occurrence and development of atrial fibrillation.