| Objective: Atrial Fibrillation(AF) is one of the most common tachyar rhythmia. The mobidity increases along the age increasing. It easily causes serious complication including atrium thromb and cerebral embolism[1-3]. The previous researches have verified that the potassium and calcium channels in cardiac myocytes changed obviously in AF, while currents and encoding genes and proteins level also displayed up-regulation or down-regulation correspondingly[4-8]. The transient outside current(Ito) is an important repolarizing k+ current in human heart, which is especially important for the early phase of repolarizaion. It controls the height of the early plateau of the action potential and thereby influences other currents that function in following phases of repolarization[9]. KChIP2(Kv channel interacting protein 2,KChIP2) is the most important auxiliary subunit of transient outside potassium channel, which plays an important role in regulating the channel gene expression and the current characteristics[10, 11]. Here, this study is to detect the KChIP2 mRNA level in AF by real-time PCR, and discuss the role of KChIP2 in AF and regulation the Ito. Methods:â‘ 30 right atrial appendage samples from patients with the rheumatic heart disease(RHD) whom accept cardopulmonary bypass(CPB) were divided two groups: the AF group (n=17) and the sinus rhythm(SR) group (n=13).â‘¡Acquire the total RNA from the atrial tissues, and the standard preparations were the reconstructed plasmids concluding the target gene by RT-PCR and TA cloning.â‘¢Compare the KChIP2 and KCND3 gene mRNA level between the two groups by SYBR Green I real-time PCR with the GAPDH as the house keeping gene. Results:â‘ the quality of the total RNA: The ratio of A260/280 was between 1.80-2.10 and the ratio of A260/230 was between 1.90-2.20 by UV spectrophotometer. From the gel electrophoresis, the 28S band and the 18S band were obvious, the gray scale of the 28S band was more than the 18S band. The 5S band was very gloomy.â‘¡the standard curve and the melting curve: the standard curves had good linear correlation, the contiguous coefficient R2>0.99. The standard curve of the KChIP2 gene was y=-0.23x+6.69(R2=0.994); The standard curve of the KCND3 gene was y=-0.20x+6.24(R2=0.996); The standard curve of the GAPDH gene was y=-0.23x+5.94(R2=0.998). The shape of the melting curves was unimodality. The Tm values of the products were very approximate to the predictive values.â‘¢The respective ratio of KChIP2/GAPDH between the two groups was 0.2200±0.0388(n=13) and 0.1468±0.0452(n=17), P<0.01; the respective ratio of KCND3/GAPDH was 0.5257±0.1427(n=13) and 0.3946±0.1826 (n=17), P<0.05; Conclusions: Compared to the RHD patients with SR, the KChIP2 and KCND3 mRNA levels in the patients with chronic AF were down-regulation significantly. The down-regulation of the KChIP2 and KCND3 gene is one of the molecular bases on the down-regulation of the Ito current density in AF. Objective: The transient outside K+ current(Ito) in the human heat is responsible for the initial phase of repolarization, which is especially important for the early phase of repolarizaion and influences other currents that function in following phases of repolarization. Lots of researches indicated that, the magnitude of Ito is reduced in various cardiac diseases, including heart failure[15], myocardial infarction and atrial fibrillation[4,5,30]. Downregulation of Ito is accompanied by a significant reduce of the gene and protein level. Downregulation of Ito and ICa-L is an important ionic mechanism of atrial electric remodeling(AER), which playes an key roles on the genesis and maintenance in AF. KChIP2 is the most important auxiliary subunits of transient outside potassium, which plays an important role in regulating the channel gene expression and the current characteristics. KChIP2 can promote the transport of the transient outside K+ channel protein from endoplasmic reticulum(ER) to cellular membrane and increase the expression of channel in membrane. Further more, it can increase the Ito density greatly, slow the inactivation, accelerate the recover from the inactivation and increase the mRNA level of the Kv4 channel, which contributes to that the electrophysiolgical characteristics is near the native current[10,11,23,24,31-33]. This study will construct the expression plasmid pEGFP-KChIP2 of the human atrial myocytes KChIP2 gene by RT-PCR and directed cloning techniques and establish the basis for researching the KChIP2 regulation function. Methods:â‘ Extract the total RNA from the right atrial appendage and acquire the cDNA through reverse transcription;â‘¡Acquire the full length of the KChIP2 CDS region by PCR amplification with specific primers and the reconstructed cloning plasmid pMD18-T-KChIP2 through subcloning with pMD18-T plasmid;â‘¢Design the specific primers including the enzyme cutting sites Hind III and BamH I; acquire the the full length of the KChIP2 CDS region concluding the enzyme cutting sites;â‘£construct the double enzyme cutting reaction containing the full length of the KChIP2 concluding the enzyme cutting sites and the pEGFP-c1 plasmid; reclaim the electrophoresis products and progress the ligated reaction;⑤The ligated products were transferred to the competence cell DH5αand were cultured overnight; extract the reconstructed plasmid for sequencing and identificating. Results:â‘ The location of the full length of the KChIP2 was coincidence with 663bp.â‘¡Compared the sequence of the reconstructed plasmid with the data from the PubMed database(AY026328), there was 661bp which was the same as the database and the homology was 99%. The mutation sites the 253bp(Tâ†'C) and the 344bp(Tâ†'C), which coding amino acids were mutated to Leucine(CTC) and Serine(TCC) from the Phenylalanine(TTC and TTT) respectively. Conclusions: the reconstructed expression plasmid was constructed successfully, which established the basis for further research on the regulation of KChIP2 for Ito. |
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