| Objective: Hyperpolarization-activated cyclic nucleotide-gated(HCN)channel is a nonselective cation channel.It was reported that only monovalent cation(Na+,K+)could pass through this channel under normal physiological conditions.In recent years,several studies have shown that the channels allows the passage of a small amount of Ca2+ into cells,which does not change the intracellular calcium homeostasis.In mammalian heart,the HCN1,HCN2 and HCN4 were expressed,however,only HCN2 and HCN4 contribute to normal physiological activity.Furthermore,the If currents were recorded successfully on the cell model expressing heterologous HCN channels.It is well-known that the HCN channels are the molecular basis of pacemaker current(If)that is thought to play an important role in the generation and autonomic regulation of the diastolic depolarization.Atrial fibrillation(AF)is one of the most malignant arrhythmia in clinical practice.Numerous studies have shown that increased expression of HCN channels in atrium contribute to the enhanced arrhythmicity in patients with AF,and If channels have been antiarrhythmic target of AF.Ivabradine is a specific HCN channel blocker,which has been used to treat chronic stable angina and arrhythmia.Clinical studies have shown that ivabradine decreases the automaticity of sinoatrial node and heart rate,but the underlying mechanism remains incompletely understood.In this study,the pacemaker model of neonatal rat atrial myocytes(NRAMs)was established,and the effect of ivabradine on the expression of HCN channels in NRAMs was investigated.Methods:(1)The preparation and culture of primary rat atrial myocytes: 1~3 days Sprague Dawley(SD)rats were selected.In sterile condition,the limbs of rats were fixed and the chest was opened.The hearts were harvested and put into P6 petri dish containing sterile PBS.The hearts were rinsed and right or left auricles were isolated under a magnifier,which were collected and transferred to T25 glass culture bottle containing 0.25% trypsin and were continued to incubate at 4?.After 14~16 h,the digestion was terminated with complete medium,which was then refreshed with the same medium containing type II collagenase for second digestion.The isolated atrial myocytes were collected and inoculated on P10 petri dishes.The cells were incubated at incubator(37? and 5% CO2)to separate myocytes from fibroblast cells.The myocytes were inoculated about 1 x 104 /cm2,and 5-BrdU(100 ?M)was added in the medium to inhibit growth of fibroblast cells.Cells were observated through the inverted microscope after incubation 24 h.(2)The NRAMs were randomly divided into 4 groups: control group;drug intervention group(Iva),10 ?M Ivabradine in the medium;pacemakergroup(Pacing),a series of continuous electric stimulation was given to cells(size of 10 V,10 Hz frequency,duration of 24 h)to simulate rapid pacing model;and pacemaker intervention group(Pacing + iva): 10 ?M ivabradine was added in the medium in addition to the same electrical stimulation.(3)Real-time PCR: total RNA was extracted according to kit instructions,and the ratio of 260/280 were all between 1.8~2.0.Total RNA was reverse-transcribed and the expression of HCN2,HCN4,AIF,microRNA-1,and microRNA-133 were evaluated by q-PCR.(4)Western blotting: total protein was extracted by RIPA including protease inhibitors,and protein concentration was measured using BCA method.The protein lysates were denaturated to make them stable.The lysates were separated by SDS-PAGE and transferred to the PVDF membrane.The PVDF membranes were incubated with antibodies and the images was analyzed by Bio-Rad Imaging System and Quantity One software.(5)Flow Cytometry(FCM)was used to evaluate cell apoptosis.(6)The whole-cell patch clamp experiment: the NRAMs were observed under inverted microscope,the cells with well stereo were selected for electrophysiology experiment.Results:(1)Real-Time PCR: 1)Compared with the control group,the Iva group exhibited significantly elevated expression of HCN2(n=6,P< 0.01)and HCN4(n=6,P< 0.05)mRNA,and the pacing group also exhibited significantly enhanced levels of HCN2(n=6,P< 0.01)and HCN4(n=6,P< 0.05)mRNA.Comparedwith the pacing group,the pacing+iva group showed significantly downregulated levels of HCN2 and HCN4 mRNA(n=6,P< 0.05).2)The Iva group exhibited significantly depressed expression of microRNA-1and microRNA-133 compared to those of the control group(n=6,P<0.01),and the pacing group also exhibited significantly decreased expression of microRNA-1 and microRNA-133(n=6,P<0.01).Compared with the pacemaker group,the pacing+iva group showed significantly enhanced levels of microRNA-1 and microRNA-133(n=6,P< 0.01).(2)Western blotting: The Iva group was demonstrated to have significantly elevated expression of HCN2 and HCN4 proteins compared with those of the control group(n=5,P< 0.01).However,the expression of AIF was not different between the two groups(n=5,P> 0.05);similarly,the pacing group also exhibited significantly enhanced HCN2 and HCN4 protein expression compared to those of the control group(n=5,P< 0.01),and the pacing group showed the higher expression of AIF than that of control group(n=5,P< 0.01);in addition,compared with the pacing group,the pacing+iva group showed significantly downregulated levels of HCN2 and HCN4 protein(n=5,P< 0.01),and the level of AIF was also suppressed in the pacing+iva group(n=5,P< 0.01).(3)Flow Cytometry(FCM):Compared to the control group,the Iva group did not show any increased apoptosis rate(n=5,P> 0.05),whereas the pacing group exhibited a significantly higher apoptosis rate(n=5,P< 0.01);morever,the pacing+iva group was observed lower percentages of apoptosis than thatof the pacemaker group(n=5,P< 0.01).(4)Patch-clamp experiments:At-150 mV of test potential,currents density of If of NRAMs was decreased from-12.49 ± 3.26 pA/pF to-4.83 ± 1.97 pA/pF by 10 ?M ivabradine(n=6,P< 0.01),and current density of If of NRAMs was increased from-12.49 ± 3.26 pA/pF to-17.32 ± 2.73 pA/pF in the pacing group(n=6,P< 0.05),compared with the pacing group,while the value of If was decreased from-17.32±2.73 pA/pF to-5.05±2.27 pA/pF by 10 ?M ivabradine(n=6,P< 0.01).Conclusions:(1)Using the trypsin combined with type II collagenase digestion method,we can get more NRAMs that are suitable for molecular biology experiments and electrophysiological study.(2)The pacemaker model on the NRAMs was established successfully.(3)HCN2 and HCN4 channels were involved in the occurrence and development of atrial fibrillation.(4)Ivabradine inhibited the expression of HCN2 and HCN4 at both mRNA and protein levels,and inhibited pacemaker current,we speculate that ivabradine has therapeutic effect on AF. |
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