Experimental Study Of Expression Of Gene Kcnj2Inhibited By Recombinant Adeno-Associated Virus(rAAV)-Mediated Rna Interference In Rat Cardiomyocytes

Background Currently electronic pacemakers ranks as the most prefered therapy for heart block and sick sinus node syndromes. But it suffers a lot of disatvantages:limited battery life, infections, reoperations and venous thromoembolus, etc. Furthermore, they are not suitable for patients that apt to infection or being too young neither. With the development of moleculerbiologial technology, people are trying to perfect the biological pacemaker. 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)Overexpressing the neu-rohormone receptors to increase the atria electronic activity.(2)Overexpressing the HCN2in diastolic phase.(3) Suppressing the inward-rectifier potassium current(Ik1) to break the balance of the potassium currenct inside the ventricular cells, which then can obtain the capability of self-discipline. The first two methods can only reach the standard of low beating rate by vagal stimulation to suppress the sinus rhythm. Then it is can be concluded that these methods are not suitable for making biological pacemaker.Recently study demonstrates that adult myocardial cell enjoys the latent pacing ability, but it is inhibited by inward rectify current (IK1). With the aid of powerful inward rectifier properties holding the rest potential at negative level, IK1then is able to inhibit the myocyte spontaneous electrical activity. IK1potassium channel is coded by gene KCNJ2, abundant in atrial and ventricular myocytes while sinus node cell is lack of this kind of currents. It is then assumed that ventricular myocyte will 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 pressure placed on IK1is, the more 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 the physiologyical 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. So, we design a experimental study of suppression of KCNJ2gene by RNA interference(RNAi) technology in order to provide some thoughts for the development of biological pacemakerRNA interference is a phenomenon of gene silencing at the lever of post transcription resulted from the degradation of mRNA reduced by double strands RNA. It was firstly founded in Caenorhabditis elegans. Recently, with the advanced performance of RNAi, people are able to reduce specific gene silencing of mammalian cells by way of siRNA synthesized in vetro or expressed through vector in vivo.RNAi is a powerful moleculerbiologial technology for knocking down genes in recent years. The suppression of RNAi to the target genes is highly specific and stable.The RNAi has an amplification effects. There are two RNAs being used in the RNAi:siRNA(small interference RNAs) and shRNA(short hairpin RNAs). The shRNA is more effective than the siRNA. The pratical programme is stepped as follows:(1)Selecting the target gene.(2)Chosing the interference target sites.(3)Selecting the methods to check the genetic functions.(4)Screening the most effectively interference site.(5)Constructing the vectors to treat gene disease. Previous study of our team has screened out the most obvious suppressing sites on KCNJ2gene mRNA, namely+361-+379bp.Recombinant adeno-associated virus (rAAV) are transformation form being replaced AAV rep and cap genes for other gene and expression components. The main advantages of rAAV as gene therapy vectors are as follow:(1)AAV is a human-derived virus and has non-pathogenic to humans. Being mild immune response, the recombinant AAV can get rid of96%of the original negative gene groups to further ensure safety.(2) Site integration can be more stable, thus avoiding not only the integration of other viruses caused by random inactivation of tumor suppressor genes but the risk of oncogene activation.(3) AAV-mediated gene expression can be sustained.(4) AAV has a wide host range, including mitosis and a variety of non-dividing cells.(5) AAV has good thermal stability and acid alkaline. Thus, AAV has become the most advantaged new vector in gene therapy.This study will give evidence for creating a biological pacemaker by the application of RNAi in rat. There is no report about creating a boilogial pacemaker by way of RNAi until now. The study contains three parts as stated in details below.Part1. Culture and viability of neonatal rat cardiomyocytes ObjectiveTo obtain the rats cardiomyocytes with high purity and viability as well as to provide the experimental model for the coming experiment. MethodsThe rat ventricular muscles were enzymaticaly dispersed by use of0.1%trpsin. The cells were purified by attachements and adding BrdU, and then identified by immunohistochemical stain. The beating rates of ten cells were recorded under phase contrast microscope by the intervals of3days,6days and9days, respectively.ResultsThe survial rate of myocytes after isolation was94.6%while the purity qualifies to95%, the viability of cells recorded after intervals of3,6and9days were not different from each other.Part2. Construction of rAAV vector rAAV2-EGFP-U6-kir2.1-shRNA ObjectiveTo construct specific rAAV to rat cardiomyocytes gene KCNJ2mRNA.MethodsAccording to previous study we have obtained the most obvious sites in the KCNJ2gene mRNA. Basing on that, vector-plasmid transfection were utilized to package cells (BHK-21cells), then cultivate cell quantity to passage up to certain level to aid virus (HSV2-rc/-UL2) in infecting packaging cells, cell lysis, chloroform extraction and purified accumulated adeno-associated virus vector rAAV2-EGFP-U6-kir2.1-shRNA.ResultsThe vector rAAV2-EGFP-U6-kir2.1-shRNA was successfully constructed by the strategy "one virus to infect one cell line" Part3. The influence on the KCNJ2gene expression and beating frequencies in rat cardiomyocytes by rAAV-conducted RNAiObjective:1. To confirm the MOI of the rAAV vector to rat cardiomyocytes.2. To observe the variation of the rat cardiomyocytes beating rates after the rAAV infection.3. To observe the alteration of the expression of KCNJ2gene and the Kir2.1protein after the rAAV infection.MethodsThe neonatal rat myocytes were divided into three groups after cultured.(1)Interference group:infected rAAV vector rAAV2-EGFP-U6-kir2.1-shRNA;(2)Negative control group:infected negative rAAV;(3) Controll group:no treatment.The suprresing rates were then calculated by RT-PCR method. The protein was detected by Western-blotResults1. The best MOI to rAAV infection in rat cardiomyocytes was fixed to1×106vp/cell.2. The beating rates of rat cardiomyocytes were increase after the rAAV infection.3. The expression of KCNJ2gene and Kir2.1protein were suppressed after the rAAV infection.