| BackgroundAtrial fibrillation (AF) is one of the most common arrhythmia with substantial morbidity, mortality, and socioeconomic burden. Experimental and clinical studies have shown that electrical remodeling (ER) and structure remodeling (SR) are two major mechanisms involved in the AF. ER occurred early in the course of AF and led to characteristic changes in action potential duration (APD) and effective refractory period (ERP), whereas SR starts later and proceeds more slowly. Recently, abnormal expression of gene encoding the ion-channel protein, especially potassium (κ) channel, has attracted the researchers’interest in the molecular mechanism for ER and arrhythmia. Also, the difference between the level of messenger ribonucleic acid (mRNA) and that of the correspondent protein, frequently observed in gene expression studies, has aroused researcher’s interest to investigate the regulatory mechanisms at the post-transcriptional level. Although traditional pharmacological therapies were effective in maintaining normal sinus rhythm, they were associated with numerous adverse side reactions such as nausea, dizziness, fatigue and even ventricular arrhythmias, while non-pharmacological therapies, such as current cardioversion, surgery, radiofrequency ablation, for instance, may lead to complications such as infection and thrombosis. Therefore, it is important to find an efficient and safe new strategy for the treatment of the AF. MicroRNAs (miRNA), a group of endogenous single-stranded non-protein-coding small RNAs (-22nucleotides long) were initially described in1993. MiRNA interacts with the3’untranslated region (3’UTR) of its target mRNA via exact complementarity with the7-8nt at its5’ end, the so-called’seed sequence’, which is critical for miRNA actions to guide RNA induced silencing complex (RISC) to down-regulate the expression of its target mRNA at the post-transcriptional level.MicroRNA-1(miR-1) was known to be muscle-specific miRNAs preferentially expressed in adult cardiac and skeletal muscle tissues, which was the top20abundant miRNAs in human heart. Current studies indicated that miR-1is involved in many heart diseases, especially in cardiac arrhythmias and its expression is associated with cardiac arrythmogenic potential in ischemic heart diseases. Delivery of miR-1into normal or infarcted rat hearts induces significantly widened QRS complex and prolonged QT interval in electrocardiograms, and AMO-1(anti-miR-1inhibitor oligonucleotides) could rescue this effect. The upregulation of miR-1could increase conduction time and depolarize the membrane potential through repressing the levels of Kir2.1and Connexin43, which may be partly responsible for its arrhythmogenic potential. Recently, one study by Terentyev et al suggested that miR-1may also participate in arrhythmia by deteriorates Ca2+handling.The miRNA of rabbit is absent in the miRbase until now. Though miR-1has been studied in the pathogenesis of cardiac arrhythmia, its possible role in the early stage of atrial ER has not yet been reported. Therefore, we performed the present study, to provide the first evidence that miR-1played a significant part in the early stage of atrial ER through a rabbit model with1-week right atrial tachypacing (A-TP). We used in this work established animal techniques for electrophysiology measurements and lentiviral vectors (LV) to deliver the genes of interest. The advantage of LV is their low immunogenicity and they can be easily transferred into cells and tissues and lead to much higher gene expression than that achieved by using adenovirus vectors in previous similar experiments.Objectives1. To evaluate the expression of miR-1in right A-TP rabbit model by a pacemaker for 1-week.2. To definite the relationship between the abnormal expression of miR-1and the early stage of atrial ER in AF.3. To elucidate the potential molecular mechanisms of miR-1accelerates the early stage of atrial ER in AF.Methods1. Establishment of animal model1.1Animal preparationAdult New Zealand White rabbits (regardless of their gender;1.5-2.5kg) were randomly allocated into4groups:1:control group (Ctl, n=6):with no pacing and transfected with control LV;2:right A-TP group (Pacing, n=6):submitted to pacing at600beats per minute (bpm) for1week, then transfected with control LV;3:right A-TP group transfected with miR-1(P+miR-1, n=6), recombinant LV carrying miR-1were injected into right atrial after right A-TP;4:right A-TP with AMO-1(P+AMO-1, n=6), where animals, after right A-TP, were injected into right atrial with recombinant LV carrying AMO-1.1.2Establishment of right atrial tachypacing (A-TP) modelRabbits were anesthetized with pentobarbital sodium (30-35mg/kg) and were ventilated by tracheostomy with a volume-regulated respirator. Halothane and N2O were supplemented to maintain a constant level of anesthesia for all procedures. Ventilator settings were adjusted to maintain physiological arterial blood gases. After administrating local anesthesia with lidocaine in the neck skin, right jugular vein was isolated and ligated by skin incision. A pacemaker was implanted in a subcutaneous pocket and attached to an electrode-lead in the RA appendage via the right jugular vein under the guidance of X-ray. All surgical procedures were performed under sterile conditions.2. Electrophysiological monitorElectrophysiological examination was performed at3time points (before pacing, before transfection and1-week after transfection), by using a programmable multichannel stimulator and intracardiac electrograms (ECG) were measured by using electrophysiological recording system, by placing the catheter into the right atrial (RA). Atrial effective refrractory period (AERP) was measured with S1-S2programmed electrical stimulation [PES]. AF was induced by PES with burst stimulation.3. Construction and production of Lentiviral vectorThe miR-1and AMO-1sequences of New Zealand White rabbit precursor were synthesized by TELEBIO (Shanghai, PR China). The titer of Lentiviral vector was1×109TU/ml.4. In vivo gene transferAfter1week of A-TP, the heart was exposed and the RAs were fixed. Then recombinant LVs were directly injected into RAs. After that, the heart was placed back into the thoracic cavity. One week after lentiviral injection, the rabbits were killed and the heart was immediately excised and washed by ice-cold PBS retroperfusion via the aortic root, then the RA was dissected, blotted dry, frozen in liquid nitrogen, and stored at-80℃.5. Real-time qRT-PCR analysisTotal RNA was extracted from rabbits RAs, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) was performed to detect the level of miR-1and KCNE1and KCNB2genes.6. Western blot analysisWestern blot analysis was used to determine the protein expression of KCNE1and KCNB2.7. Luciferase reporter assaysKCNE1and-KCNB2as the target genes for miR-1were confirmed by luciferase activity assay.Results1. The early stage of atrial ER in AF was found in right A-TP rabbit model by a pacemaker for1-week; 2. The expression of miR-1was increased with the right atrial ER;3. Over-expression of miR-1via in vivo transfections of recombinant LV accelerated right atrial ER after A-TP meanwhile AMO-1could rescue it;4. The level of miR-1and KCNE1/KCNB2appear to be negatively correlated, indicating thus that miR-1could accelerate the atrial ER through KCNE1and KCNB2target genes.ConclusionsMiR-1accelerates the atrial ER induced by A-TP via target K+channel genes, indicating that miR-1may play an important role in the initiation of AF. Thus, miR-1has great clinical relevance as a potential therapeutic target for AF. |
PDF Full Text Request