| Objective:Hyperpolarization activated cyclic nucleotide gated channel(HCN)is a family of genes which has been widely concerned since its discovery.The main function of HCN channel is to encode hyperpolarization activated cation current(if).The current plays an important role in the physiological activities of various organs,especially in the electrophysiological activities of sinoatrial node.HCN channels are composed of homologous or non homologous tetramers,forming different subtypes of HCN1,HCN2,HCN3and HCN4.HCN widely exists in heart,nerve,gastrointestinal,bladder and other tissues and organs.In electrophysiological activities,HCN1 was activated the fastest,followed by HCN2 and HCN3,and HCN4 was the slowest.The molecular structure of HCN channel is a transmembrane protein,which is composed of six transmembrane helix segments,COOH terminal and NH2terminal three important modules.It has dual characteristics of voltage-gated and chemical gated.K~+and Na~+can pass through the channel,and cyclic adenosine monophosphate(c AMP)also affects its activity.At present,many cardiovascular diseases have been found to be associated with abnormal HCN channels.When HCN channel protein gene mutation reduces HCN density,it can cause a series of bradyarrhythmias,such as hereditary sick sinus syndrome;basic research and clinical experiments on atrial fibrillation have confirmed that the expression of HCN channel in animal and human sinus node cells is up-regulated;in the study of myocardial diseases,the expression of HCN channel in ventricular muscle is also up-regulated.Clinical often see a variety of factors lead to cardiac hypertrophy patients,in this pathological state,the heart often occurs ventricular arrhythmia,which increases the risk of sudden cardiac death in patients with cardiovascular disease.Previous studies have shown that the expression of HCN channel in ventricular muscle is increased during myocardial hypertrophy.Therefore,we speculate that the up regulation of HCN expression delays the repolarization time of ventricular action potential,that is,the QT interval is prolonged,resulting in the increase of ventricular excitability and arrhythmia.In addition,cardiac hypertrophy is often accompanied by the increase of sympathetic nerve activity,which further aggravates the occurrence of arrhythmia and even fatal arrhythmia.Ivabradine is a highly selective and specific inhibitor of HCN channel.Recent studies have shown that ivabradine has other effects in addition to reducing sinus heart rate:ivabradine can reduce the infarct size in the animal model of ischemia-reperfusion injury by inhibiting the production of mitochondrial reactive oxygen species,and ivabradine can also inhibit the formation of mitochondrial reactive oxygen species Inflammatory response,reduce the indicators of vascular oxidative stress,reduce the formation of atherosclerotic plaque and anti fibrosis in apolipoprotein E(Apo E)deficiency mice.However,the research data on the effect of ivabradine on sympathetic nerve is still insufficient,so this experiment is to verify the expression of HCN2 and HCN4channels in the ventricular muscle of rats with myocardial hypertrophy and the inhibitory effect of ivabradine on them,and to explore the potential effect of ivabradine on sympathetic nerve.Methods:1.Animal model establishment:Eighteen adult rats,SD,SPF,male,weighing 260-300g,were randomly divided into three groups:the sham group(Sham),the model group(Model)and the ivabradine group(IVA),with 6rats in each group.Rats in the model group and ivabradine group were treated with abdominal aortic coarctation,while rats in the sham operation group were operated as before,but without ligation.12 weeks after operation,ivabradine group was given ivabradine hydrochloride by gavage,the dose was 10mg/(kg·d)according to the relevant studies.The sham group and model group were given the same volume of normal saline by gavage,and fed for 5weeks.2.Calculate HW/BW and LVW/BW:Before the rats were killed,the body weight(BW)of all rats were weighed,and then the hearts were separated,washed with PBS liquid,squeezed and dried with filter paper.After the residual liquid was drained,the whole heart was put on the scale,and the heart weight(HW)was read.Then the left ventricular tissue was separated and put on the scale to read the left ventricular weight(LVW).Finally,the whole heart mass index(HW/BW)and left ventricular mass index(LVW/BW)were calculated.3.Immunohistochemistry(IHC)and HE staining:A little apical tissue and left ventricular tissue were cut and put into 4%paraformaldehyde solution respectively.Apical tissue was used for HE staining to detect the pathological changes of hypertrophic myocardium,and left ventricular tissue was used for IHC staining to detect the expression of tyrosine hydroxylase(TH)in myocardial tissue to show the distribution of sympathetic nerve.4.Immunohistochemical test(ELISA):After thoracotomy,3ml of left ventricular blood was obtained by acupuncturing the blood sample into the apex of heart.The blood sample was placed aside,centrifuged 30min later(rotating speed was 3000r,time was 15min),and the separated serum was transferred into a 1.5ml EP tube with a pipette and stored in a refrigerator at-80℃.Then,the heart was isolated,and part of the left ventricular tissue was cut and put into EP tube,and put into the refrigerator at-80℃.Finally,the contents of noradrenalin(NA)and epinephrine(EPI)in serum and myocardial tissue of rats were detected by immunohistochemistry.5.Western blot(WB):The protein expression of HCN2 and HCN4 channels in myocardium was tested by Western blot.6.Quantitative polymerase chain reaction(q PCR):Total RNA was extracted from rat ventricular septal myocardium,and the m RNA expression of HCN2 and HCN4 channels in myocardium was determined by q PCR.Results:(1)Comparison of myocardial mass index:the HW/BW and LVW/BW of the Sham group were 0.26±0.02 and 0.19±0.02,the HW/BW and LVW/BW of the Model group were 0.30±0.03 and 0.22±0.04,the HW/BW and LVW/BW of the IVA group were 0.32±0.02 and 0.22±0.02,compared with the sham group,myocardial mass index of the model group and the IVA group were increased,P<0.05;but compared with the model group,myocardial mass index of the IVA group had no significant change,P>0.05.(2)HE staining:Compared with the sham group,cardiomyocytes in model group grew longer,nuclear volume increased and arranged sparsely and irregularly;compared with the model group,cardiomyocytes in IVA group arranged regularly.(3)IHC staining:(1)HCN channel:in sham group,HCN2(256.7±50.95)and HCN4(2554.69±394.7)were slightly expressed in ventricular myocytes;compared with sham group,HCN2(3500.17±456.53)and HCN4(5886.36±550.774)in model group were increased,P<0.05;compared with model group,HCN2(1477.78±279.53)and HCN4(4718.63±713.52)in IVA group were relatively decreased,P<0.05.(2)Sympathetic nerve(TH):the content of TH in sham group was 167.94±30.65,compared with it,the content of TH in model group which was 1996.22±382.45 increased,P<0.05;compared with model group,the content of TH in IVA group which was 745.3±143.36 decreased,P<0.05.(4)The results of immunohistochemistry were compared between groups:norepinephrine in serum and in tissue of sham group were 4.17±1.09ng/ml and 16.98±3.44ng/ml,compared with sham group,norepinephrine in serum and in tissue of model group which were 12.55±2.29ng/ml and 37.02±5.75ng/ml,were higher,P<0.01.In relation to model group,norepinephrine in serum and in tissue of IVA group which were 4.93±1.71ng/ml and 23.75±5.72ng/ml,were decreased,P<0.01.EPI in serum and in tissue of sham group were 1.3±0.39ng/ml and 7.69±1.05ng/ml,compared with sham group,serum EPI(5.42±0.66ng/ml)and tissue EPI(13.8±1.67ng/ml)were increased in model group,P<0.01.Compared with model group,serum EPI(2.62±0.61ng/ml)and tissue EPI(9.58±1.99 ng/ml)were decreased in IVA group,P<0.001.(5)Inter group western blot test:In sham group,the level of HCN2 and HCN4 protein were 0.17±0.08 and 0.13±0.04,compared to it,the level of HCN2(0.60±0.07)and HCN4 protein(0.65±0.06)were increased in model group,P<0.001.Compared with model group,the level of HCN2(0.37±0.03)and HCN4 protein(0.32±0.07)were decreased in IVA group,P<0.001.(6)Comparison of quantitative polymerase chain reaction results:the level of HCN2 m RNA and HCN4 m RNA were 1.13±0.22 and 0.09±0.33in sham group,compared with it,the level of HCN2 m RNA(4.45±0.60)and HCN4 m RNA(4.46±0.60)were increased in model group,P<0.001;compared with model group,the level of HCN2 m RNA(2.48±0.19)and HCN4 m RNA(2.5±0.31)were decreased in IVA group,P<0.001.Conclusion:1.Both HCN2 and HCN4 channels are expressed in normal rat ventricular muscle.2.The expression of HCN2 and HCN4 channels increased in ventricular myocytes of hypertrophic rats,and the expression of sympathetic nerve in systemic system and myocardial tissue also increased.3.Ivabradine can inhibit the expression of HCN2,HCN4 channels and sympathetic nerve in ventricular muscle of rats with myocardial hypertrophy. |
PDF Full Text Request