Down-Regulation Of Nkx2.5 Reprogrammes Rat Cardiomyocytes Into Pacemaker-Like Cells

PART Ⅰ Down-regulation of Nkx2.5 reprogrammes neonatal rat cardiomyocytes into pacemaker-like cellsOBJECTIVE: To investigate whether down-regulation of Nkx2.5 by adenovirus-mediated short hairpin RNA can reprogramm neonatal rat cardiomyocytes into pacemaker-like cells in vitro.METHODS: Neonatal rat cardiomyocytes were digested and isolated by trypsin and type II collagenase.The shape of cells were observed by using the opitical microscope.Immunofluorescopy was used to detect the purity.Randomly divided into negative control group and experimental group.They were transfected with recombinant adenovirus carrying sh RNA targeting Nkx2.5 and expressing green fluorescent protein(GFP),Ad-Nkx2.5-sh RNA-GFP,and the control adenovirus expressing GFP only,Ad-sh RNA-GFP,at different multiplicities of infection(MOI)in order to select the most suitable MOI.After transfection for 48 h,beating frequency of cells were counted.The silencing effect of Nkx2.5,the levels of pacemaker-related transcription factors(Tbx18,Tbx3,Shox2,ISL1),hyperpolarization activated cyclic nucleotide gated potassium channel 4(Hcn4),and connexin 40(Cx40))were measured by real-time PCR and Western blot.Immunofluorescopy was used to detect the expression of HCN4 channels.After 72 hours of transfection,the cells were detected the If current of green fluorescent cells by using whole-cell patch clamp.RESULTS: After 48 h,Neonatal rat cardiomyocytes isolated and cultured showed cluster-like beatings.The purity of cardiomyocytes were up to 0.91±0.01 by the detection of α-actin.The most suitable MOI was 20.The Ad-Nkx2.5-sh RNA-GFP was successfully transfected into cardiomyocytes in vitro,and significantly down-regulated the expression of Nkx2.5 at m RNA and protein levels(p<0.05);Cells generated synchronous beat,the beating rates in negative control group and experimental group were 118.8±1.3bpm vs 138.7±4.0 bpm;after down-regulation of Nkx2.5 in cardiomyocytes,the levels of pacemaker-related transcription factors and HCN4 expression were enhanced(p<0.05);The expression of Cx40 was decreased(p<0.05).Fluorescence microscopy showed that the red fluorescence of the experimental group,which reflects the expression of HCN4 channel,were stronger than that of the negative control group.After 72 hours of adenovirus transfection,the green fluorescent cells in the experimental group were detected If current that can be completely blocked by specific blocker CsCl.Conclusion: Down-regulation of Nkx2.5 in neonatal rat cardiomyocytes can increase the levels of transcription factors of pacemaker-cells and HCN4 channel,decrease the level of Cx40,and detect If current,which indicates that down-regulation of Nkx2.5 gene can reprogramm neonatal rat cardiomyocytes into pacemaker-like cells.Part Ⅱ The effect on ventricular rate caused by down-regulation of Nkx2.5 in rats with complete atrioventricular block modelOBJECTIVE: To investigate the effect on ventricular rate caused by down-regulation of Nkx2.5 in rats with complete atrioventricular block model.METHODS: Twenty adult Sprague-Dawley rats were randomly divided into the control group(n=10)and experiment group(n=10).Adenoviruses were injected into the left ventricular apex of rats.After lateral thoracotomy,a needle was inserted at the free wall apex of the left ventricle.50 μl of adenovirus containing 8x108fluorescence-forming units of Ad-sh RNA-GFP or Ad-Nkx2.5-sh RNA-GFP was injected into the left ventricle apex.At 7 days post-transplantation,the hearts were harvested and perfused using a Langendorff apparatus and recorded electrophysiological datas.Injecting 75% ethanol to ablate the atrioventricular node of Koch triangle to prepare a complete atrioventricular block model.Electrode-pacing was performed at the site of transgene injection,comparing the difference between spontaneous pulsation of the ventricle and the pacing of the injection site.Observe the virus transfection efficiency of the injection site under the fluorescence microscope and confirm the Nkx2.5 silencing effect by Western blot.RESULTS: Complete atrioventricular block was observed in two groups of rats by injecting 75% ethanol to ablate the atrioventricular node.Electrocardiographic recordings of the beating hearts revealed ectopic ventricular beats in 7 of 10 hearts injected Ad-Nkx2.5-sh RNA-GFP,the average ventricular rates were 114.7 ± 3.0 bpm,and the polarity and morphology of the ectopic beats was identical to that of electrode-paced beats at the location of transgene injection,which can link the origin of the ectopic beats to the site of transduction.Conversely,the control hearts that injected Ad-sh RNA-GFP showed junctional escape rhythms at average rates of 84.5± 2.5bpm(n =10),which are not originated from the site of transduction.The injection site showed that the virus could infect the local myocardium with high efficiency by observeing under the fluorescence microscope,and effectively down-regulate the level of Nkx2.5 in cardiomyocytes of the experiment group.Conclusion: Down-regulation of Nkx2.5 level can increase the ventricular rates in rats with complete atrioventricular block.