Silencing GIRK4 Expression In Human Atrial Myocytes By Adenovirus-delivered Small Hairpin RNA

Background and objective :RNA interference by short double stranded RNA represents an efficient and frequently used way to study gene function. RNA interference utilizes sequence-specific double-stranded small interfering RNA to silence gene expression in mammalian cells. RNA interference in human cardiomyocytes is desirable as it is more persuadable to study clinic problems. Thus, gene silencing experiments in human cardiomyocytes may accelerate and increase the value for applications. Lacking of enough specimens to accomplish experiments, RNAi experiments were seldom performed in human cardiomyocytes.Cardiac I_KACh plays a major role in the parasympathetic regulation of heart rate. It is also crucial in the generation and maintenance of atrial fibrillation. I_KACh is present in the sinoatrial node, atria, atrioventricular node, and possibly Purkinje fibers of mammalian heart. I_KACh is composed of two homologous proteins:GIRK1 and GIRK4. GIRK4 is the functional subunit of I_KACh and ablation of GIRK4 should result in the functional elimination of I_KACh.GIRK1 could not form a functional ion channel without GIRK4. Moreover, the protein level of GIRK1 decreased significantly in atrial myocytes of GIRK4-knockout mice. Cardiac I_KACh was activated by various receptors coupled to G-proteins of pertussis toxin-sensitive class. Among these receptors, muscarinic 2 acetylcholine receptor was the most important one.Because I_KACh is a new therapeutic target in arrhythmia, it is necessary to further explore GIRK4 in human atrial myocytes. In this article, we explored the feasibility and problems of silencing GIRK4 in human atrial myocytes and studied the affection on GIRK1, M2AChR and I_KACh .Methods In this study, cultured human cardiac myocytes were used as experiment models. RNA interference, plasmid construct,rearrangement adenovirus, RT-PCR , western blotting and electrophysiological recording were applied to discuss the feasibility ,problems and results of silencing GIRK4.Results (1) Human atrial myocytes were cultured successfully.(2) A high effective RNAi sequence(GGCTGAGCAGAATGAAGAA) for GIRK4 was selected. (3) The GIRK4-shRNA expression plasmid(pGCsi-GIRK4-shRNA-U6) was constructed successfully. (4) The adenoviral vector for GIRK4-shRNA(Ad-GIRK4-shRNA) was constructed successfully. (5) The knockdown effiency for GIRK4 was different between the exongenous and endogenous.It was 48.9% 94.3% respectively in the exongenous and endogenous expression system.(6) RNA interference for GIRK4 were time-dependent and dose-dependent. The knockdown efficiency comparing with adenovirus-delivered nonsense shRNA group in the protein level was 15.1%, 35.2% and 51.1% respectively in MOI 5,20,50. The knockdown efficiency in mRNA level was 49.1%, 85.3%, 1.7% respectively in 24h,48h,96h. (7)Adenovirus was a sutiable vector for transferring shRNA into human atrial myocytes and the ransfection efficiency was more than 95%. (8) Silencing human GIRK4 decreased GIRK1 and increased. M2AChR Compared with the adenovirus-delivered nonsense shRNA group, GIRK1-mRNA and GIRK1 protein decreased 27.9%,20.2% respectively in Ad-GIRK4-shRNA group (0.261±0.029vs 0.362±0.038,p<0.05; 0.305±0.027vs 0.382±0.035,p<0.05). The M2AChR-mRNA and M2AChR protein increased 19.6%,18.6% respectively in sh-GIRK4 group (0.609±0.027vs 0.509±0.028,p<0.05; 0.531±0.022vs 0.448±0.023,p<0.05). (9) Silencing GIRK4 decreased the density of I_KACh. In comparison with adenovirus-delivered nonsense shRNA group, current density of I_KACh decreased 53% in Ad-GIRK4-shRNA group (–0.93±0.28pA/pF (n = 5) versus–1.95±0.51pA/pF (n = 5)respectively (p<0.01)Conclusion: we used Adenovirus-delivered short hairpin RNA targeting GIRK4 not only decreased the expression of GIRK4 in human atrial myocytes but also affected the expression of GIRK1,the expression of M2AChR and the amplitude of I_KACh.GIRK4 plays a important role in the pathway(Ach-M2AChR-G protein- I_KACh) and I_KACh channel.But there are many barriers to be overcome, if we want to acquire more effective and persistent RNAi effects in human atrial myocytes and use adenovirus-delivered small hairpin RNA against GIRK4 in clinic.