MicroRNA-499 Inhibit Cardiomyocytes Apoptosis In Pacing-induced Heart Failure In Rats

Objective:Rapid right ventricular pacing(RVP)is a classical method for studying the mechanism of congestive heart failure(CHF).At present,most of the animals used to make such models are large animals.Myocardial apoptosis is a leading cause of heart failure(HF).The aim of this study was to establish a rapid pacing induced heart failurerat model and explore the protective effect of MicroRNA-499(MiR-499)against myocardial apoptosis in non-ischemic heart failure.Methods:(1)Establishment of heart failure induced by rapid right ventricular pacing in the rat:30 adult male Wister rats with weights of 290-310 g were randomly divided into three groups:pacing group(n=10),control group(n=10)and sham group(n= 10).Pacing group rats and sham group rats underwent a pacemaker implantation surgery.Under the anesthesia of pentobarbital(60 mg/kg,intraperitoneally)and intubation of the trachea,an abdominal median incision was made,then a pacemaker was fixed in the abdominal cavity.The thoracic cavity was opened through another incision in the 4th intercostal space.The heart was carefully exposed,and an electrode was sutured to the left ventricular apex.After 7 days recovery,Sham and control group rats were reared routinely and Pacing group rats underwent rapid pacing with 550 beats/min for the 4 weeks.Then The general conditions of rats were observed and their autonomous heart rate,weight and heart mass were measured.Echocardiograms were performed to identify cardiac systolic function with the following parameters:left ventricular ejection fraction(LVEF),left ventricular percent fractional shortening(LVFS),left ventricular end-diastolic pressure(LVEDP),left ventricular end-systolic pressure(LVESP).(2)MiR-499 sequences were cloned into a recombinant adeno-associated viral vector,pAAV-NC were constructed as a negative control,then rats were infected by recombinant adeno-associated virus.Enhancing of miR-499 expression was quantified via quantitative reverse transcription polymerase chain reaction(Quantitative Real-time PCR,qRT-PCR).40 adult male Wister rats with weights of 290-310 g were randomly divided into four groups:1.Sham group(n=10),2.sham+miR-499 group(n=10),3.Pacing + NC group(n=10),4.pacing + miR-499 group(n=10).Pacing group rats and sham group rats underwent a pacemaker implantation surgery(The procedure was the same as above).After the thoracic cavity was opened and the heart was carefully exposed.AAV(5 × 1010 virus genome copies per animal)was injected into left anterior descending coronary artery during pacemaker implantation surgery of the rat of groups with miR-499 up-regulated(group 2,4).After the operation sham group rats(group 1,2)were reared routinely and Pacing group rats(group 3,4)underwent rapid pacing with 550 beats/min for the 4 weeks.The expression level of miR-499 was detected by qPT-PCR,Protein concentrations of Pscd4 and PACS2 were determined using western blot method.Apoptosis was detected via immunohistochemistry and TUNEL assay.Results:(1)The animals in the pacing group showed decreased activity,anorexia,and shortness of breath.Compared with the normal control group and sham group,the weight,LVEF and LVFS of the pacing group decreased significantly,while the autonomic heart rate,heart mass and LVEDP increased significantly(the difference was statistically significant).Pathological staining showed that the myocardial edema,the degeneration of some myocardial cells,the congestion of the interstitium,the infiltration of inflammmatory cells and the disorder of the arrangement of the fibersin the pacing group,and the fibrosis in pacing was more serious than that in control group.(2)Adeno-associated viral miR-499 was successfully transfected,and led to specific overexpression of miR-499.It isfound that enhancing the expression of miR-499 could significantly inhibit the expression of PDCD4 and PACS2 in pacing-induced heart failure rats.Furthermore,cell apoptosis was increased by pacing,but attenuated by up-regulated miR-499.Conclusion:(1)The SD rat model of CHF induced by RVP was successfully established in this study.(2)miR-499 may inhibit apoptosis in pacing-induced heart failure via PDCD4 and PACS2 pathway.