Effects Of Angiotensin ? On The Regulation Of Small Conductance Calcium Activated Potassium Channels In Human Patients With Atrial Fibrillation

bjective: Atrial fibrillation is a common clinical rapid atrial arrhythmia.A variety of heart disease can lead to the occurrence of AF.Heart damage,Ventricles rhythm?rate?disorder,atrial wall thrombosis can be caused by atrial fibrillation.Severe cases can lead to pulmonary embolism,cerebral stroke morbidity and mortality.The pathogenesis of AF is very complicated,which leads to the poor therapeutic effect.Understanding the mechanism of AF will provide a strong theoretical basis for the treatment of AF.Atrial electrical remodeling plays an important role in AF,which is characterized by the shortening of action potential duration and the decrease of frequency dependence.The duration of action potential is determined by the activity of various ion channels of the cardiomyocyte membrane,Ion channels are potential targets for the prevention and treatment of atrial fibrillation.Small Conductance Ca²⁺-activated K⁺ channels?SK?are widely expressed on cardiomyocytes.Its expression levels in atrial were significantly higher than that in ventricle.The channel is insensitive to changes in membrane potential,but it is highly sensitive to intracellular calcium.Studies have shown that the enhancement of RAS activity is closely related to the occurrence of atrial fibrillation.Inhibiting the role of endogenous angiotensin ??Ang ??can prevent the shortening of the atrial effective refractory period caused by canine atrial rapid pacing.Studies have shown that Ang ? is involved in a variety ofion channels in the electrical remodeling process,Ang ? may also cause intracellular calcium overload.We hypothesize that SK current can be changed by Ang ?,which may be involved in the electrical remodeling of AF.However,whether Ang ? has a regulatory role in SK,and its possible mechanism of action is unclear.In this study,we measured the gene expression level and protein expression level with real-time quantitative PCR method and western blotting method.SK channel current was recored by whole-cell patch clamp technique.To explore the changes of SK channel during AF and the effect of Ang ? on SK channel,by which can provide new ideas for the current treatment strategy of atrial fibrillation.Methods :?1?Electrophysiological experiment 10 cases of surgical patients with myocardial tissue in accordance with the results of ECG and medical records were divided into SR group?n=4?and AF group?n=6?.We geted single atrial myocyte from an improved method for isolation and recorded SK channel currents with whole-cell patch-clamp recording mode.Observed content:?a?The basic characteristics of SK channel in human atrial myocytes;?b?Comparison of SK channel current density and its percentage of mixed inward currents potassium currents between SR group and AF group;?c?Effect of Ang ? on SK channel current in SR group and AF group.?2?Real-time PCR experiment The AF group?n=10?and the SR group?n=10?.The expression levels of AT1 R and AT₂ R m RNA were measured in both groups.?3?Western blotting experiment The AF group?n=13?and the SR group?n=13?.The expression levels of AT1 R and AT₂ R protein expression were measured in both groups.Results:?1?The basic characteristics of SK channel in humanatrial myocytes.SK channel currents present inwardly rectifying.Apamin could specifically block the channel.?2?Compared SK channel currents between the two groups At voltages range from-130 m V to-80 m V,there was significant difference in the current density of SK channels between the two groups.At voltage-130 m V,the current density in SR group was?-2.17 ± 0.16?p A/p F and that in AF group was?-9.26 ± 0.43?p A/p F?n SR = 4,n AF = 3,p<0.05?;SK channel current in the proportion of mixed current in SR group was?29.36 ±2.8?% and that in AF group was?45.35 ± 4.3?%?n SR = 4,n AF =3,p<0.05?.The results showed that the AF group was more than the SR group,whether it was the SK channel current density or the proportion of the mixed current.?3?Ang ? regulated SK channel current from the two groups 1 ?mol/L Ang ? reduced the current density of SK channels and their proportions in the mixed current.At voltage-130 m V,the inhibition ratio of Ang ? to SK channel in SR group was?22.06 ± 1.6?% and that in AF group was?40.07 ± 1.3?%?n SR =4,n AF=5,p<0.05?.The results showed that inhibition of Ang ? in AF group was larger than that in SR group.?4?The m RNA change of the AT1 R,AT2R gene in the two groups AT1 R 2^-??Ct in SR group was?0.75 ± 0.28?,AT1 R 2^-??Ct in AF group was?0.50 ± 0.29??n SR=5,n AF=5,p>0.05?.The results showed that there was no significant difference in the level of AT1 R m RNA between SR and AF group;AT2R 2^-??Ct in SR group was?1.99 ± 0.56?,AT₂ R 2^-??Ct in AF group was?0.73 ± 0.49??n SR=5,n AF=5,p<0.01?.The results showed that AT₂ R m RNA in patients with AF cardiomyocytes reduced.?5?The relative protein expression change of the AT1 R,AT2R in the two groups The relative proteinexpression level of AT1 R in SR group was?0.22 ± 0.08?and that in AF group was?0.13 ± 0.10??n SR =6,n AF =6,p>0.05?,The results showed that there was no significant difference in the relative protein expression level of the two groups;The relative protein expression level of AT₂ R in SR group was?0.51 ±0.12?and that in AF group was?0.19 ± 0.16??n SR =7,n AF =7,p<0.05?.The results showed that AT₂ R m RNA in patients with AF cardiomyocytes reduced.Conclusions:?1?This study records the SK channel current,confirmed that human atrial myocytes exist SK channel.?2?Patients with atrial myocytes may develop an increase in calcium concentration,resulting in an increase in SK channel current density.?3?In patients with AF,Ang ? increases more prone to complex polarized terminal membrane potential shock caused by arrhythmia.