Experimental Study On The Ventriculer Rate Change Caused By Inhibition Of KCNJ2GENE Expresion With RNAi Technigue In Rat Ⅲ°AVB Model

BACKGROUNDArtificial electronic cardiac pacemaker is an electronic instrument implanting into the inside of the body.The electrical pulse produced by pulse generator which stimulating the adjacent myocardial cells, it make the heart exciting and contraction, so as to achieve treatment for some arrhythmia (mainly bradycardiac arrhythmia). Since the first cardiac pacemaker implantation in the human body in1958, it has gradually become the preferred treatment of slow arrhythmia such as sick sinus syndrome and severe atrioventricular block. With the pacemaker continuous improvement, its clinical indications is extended continuously, which gradually began to apply to the tachyarrhythmia and non electrical diseases, such as preventing paroxysmal atrial tachyarrhythmia, carotid sinus syncope, refractory congestive heart failure by biventricular pacing, etc. But in the process of its application some defects and problems have become obvious, such as limited battery life, infections, reoperations and venous thromoembolus,etc. Furthermore, they are not suitable for patients that apt to infection or being too young. With the progress of molecular biology in recent years, people try to take advantage of life science and technology research to develop a biological pacemaker to replace electronic pacemaker in order to overcome the above problems. The new technigue could repair or replace the native cardic pacemaker and the damaged conduction tissue to restore the heart pacemaker and conduction function.At present, there are two major strategies in developing biological pacemaker:gene therapy and cell therapy. Cell therapy is definited as the method of applying stem cells (embryonic stem cells and mesenchymal stem cells) and sinus node cells. But there are lots of unsolved problems such as:immunne rejection, oriented differation, tumorigenicity and ethinic issue, etc. Developing biological pacemaker by gene therapy is rooted on three strategies:(1) Over-expressing the neurohormone receptors to increase the atria electric activity.(2)Over-expressing the HCN2in diastolic phase.(3) Suppressing the inward-rectifier potassium current(Ikl) to break the balance of the potassium currenct inside the ventricular cells, which then can obtain the capability of automatic rhythmicity.Myocardium is a muscle tissue that is composed of myocardial cells.The generalized myocardial cells not only included atrial and ventricular muscle but also the S-A node,internodal bundle, atrioventricular bundle, atrioventricular junction area and Purkinje fibers. The previously study demonstrates that adult myocardial cells possess the latent of pacing ability, which it is inhibited by IK1. With the aid of powerful inward rectifier properties holding the rest potential at negative level, IK1then is able to inhibit the myocyte spontaneous depolarization. IK1potassium is coded by gene KCNJ2, abundant in atrial and ventricular myocytes while sinus node cell is lack of this kind of channel. It is then assumed that ventricular myocyte can be changed to pacemaker cell if the IK1is inhibited. Silva J and Rudy Y found that after suppression Ikl by81%, the ventricular myocytes will generate a spontaneous action; and the more on IK1is inhibated, the higher the pacing rates of myocytes is. In2002, Miake reported a biological pacemaker created by dominant-negative therapy. They got spontaneous ventricular rhythm which was more rapid than that caused by the native sinus pacemaker. The beating rates of myocytes can also respond positively to β receptors agonist, creating rudiment for the future improvment of biological pacemaking. However, in the phenotype of completely lack of IK1, the mice bears Anderson’s syndroms, such as QT-prolongatioin, periodic paralysis, skeletal and craniofacial abnormalities.RNA interference (RNAi) is a phenomenon of gene silencing at the lever of post transcription resulted from the degradation of mRNA reduced by double strands RNA.RNAi was firstly founded in Caenorhabditis elegans. Recently,with the deepening of the research on RNAi, people have been able to induce specific gene silencing of mammalian cells by way of artificial siRNA (small interference RNA) synthesized according to the principle of RNAi in vitro or expressed through vector in vivo.Lentiviral vector was transformated on the basis of HIV-1. Lentiviral vector could mediate exogenous gene expression in host cell sustained.It has many advantages, such as wide host range, more stable, accommodate bigger exogenous gene fragment, high security and so on. Thus, the lentiviral vector has become the most used vector in gene therapy.In our previous studies, we has screened out the most significant suppressing sites on KCNJ2gene mRNA,namely+361-+379bp;and found it is feasible to increasethe heart rate by using RNAi to knock down the KCNJ2targeting gene in vitro.In this study we want to use RNA interference to knock down the KCNJ2targeting gene in vivo.We observed the effect of siRNAs transfection to inhibit the KCNJ2gene expression on ventricular rate in the Ⅲ°AVB rat model, so as to provide a new effective idea and approach for the study of biological pacemaker.Part1. Construction of lentiviral vector with the shRNA effect to KCNJ2ObjectiveTo construct specific lentiviral vector with the shRNA effect to rat cardiomyocytes gene KCNJ2mRNA.MethodAccording to our previous research, the double-stranded DNA oligo which can cause most RNAi effect to KCNJ2was made. The small hairpin RNA(shRNA) sequences were annealed and linked with linearized into the lentiviral vector. Moreover, the recombinant lentivirus was harvested from293T cells when it cotransfected with lentiviral packing materials into them after72h. The virus particles were collected. Virus titer was determined by hole dilution method.ResultThe shRNA sequences were successfully inserted into lentiviral vector by double restriction digestion, and the sequences were identified by DNA sequencing. The shRNA of KCNJ2gene of the recombinant lentiviral vector was successfully packed into293T cells. The recombinant lentivirus was harvested from293T cells, and the titer of the virus of1.07x109TU/ml.conclusionThe lentiviral vector with the shRNA effect to rat cardiomyocytes gene KCNJ2mRNA was successfully constructed.Part2. Experimental study on inhibition of ventricular Ikl by RNA interference targeting the KCNJ2gene in three degree atrioventricular block ratObjective1.To establish a stable rat three degree atrioventricular block(Ⅲ°AVB) model;2.To confirm the best virus titer to lentiviral vector infection in rat;3.To observe the variation of the heart rate(HR)after the lentiviral vector infection;4.To observe the alteration of the expression of KCNJ2gene and the Kir2.1protein after the lentiviral vector infection.Method1.A50ul needle was used to inject the solutions into the myocardium toward the nodal tissue,When the insertion of the needle resulted in momentary complete AV block,25ul of70%ethanol were injected. Hearts were reinjected with ethanol if the heart block resolved after30min. After72hours, the Ⅲ°AVB was defined as stable;2.The rats were infected by lentiviral vector of different virus titer; we drawn material at different time and made frozen sections. the infection rate was detected by immunofluoresc-ence microscopy, then confirmed the best virus titer to lentiviral vector infection in rat;3.The Ⅲ°AVB rat models were divided into three groups (1) Interference group: infected lentiviral vector with shRNA;(2)Negative control group:infected negative lentiviral vector;(3) Controll group:no treatment.The HR was analyzed with electrical cardiogram;4.The levels of target genes mRNA and protein were analyzed with the real time quantitative (RT-PCR), Western blot, Immunity histochemistry.Result1.the Ⅲ°AVB rat model was stable;2.The best virus titer to lentiviral vector infection in rat was1×10TU/ml;3.The HR of rat was increase after the lentiviral vector infection;The expression of KCNJ2gene and Kir2.1protein were suppressed after the lentiviral vector infection.ConclusionIt is feasible to increase the HR by using RNAi to knock down the KCNJ2targeting gene in vivo.