Zfhx3 decreases expression of cardiac Nav1.5 channels and inhibits sodium current
Ricardo CaballeroAnabel Cámara-ChecaMarcos Rubio-AlarcónTeresa Crespo-GarcíaMaría DagoPaloma Nieto-MarínMaría José MarínRafael Salguero‐BodesJorge ToqueroJuan TamargoJorge CebriánEva Delpón
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Abstract Background Zfhx3 (zinc finger homeobox 3) is a transcription factor (TF) encoded by the ZFHX3 gene. GWAS and gene-based association studies showed that ZFHX3 is one of the major atrial fibrillation (AF) susceptibility-conferring genes. The sodium current (INa), carried by Nav1.5 channels encoded by SCN5A, is responsible for atrial and ventricular action potential depolarization and determines cardiac excitability. Zfhx3 interacts with other TFs, such as Tbx5 and Pitx2c that increase and decrease INa, respectively. However, the effects of Zfhx3 on cardiac INa are currently unknown. Purpose We aimed to determine the effects of Zfhx3 on the INa on HL-1 cardiomyocytes. Methods cDNAs encoding human Zfhx3 together or not with Pitx2c or Tbx5 were transfected in HL-1 cells. Endogenous Zfhx3 expression in HL-1 cells was silenced by means of siRNAs. INa was recorded at room temperature using the whole-cell patch-clamp and luciferase reporter assays, qPCR and Western-blot (WB) analyses were also conducted. Results Expression analysis of RNA-seq data from human ventricular (n=432) samples (GTEx) demonstrated that Zfhx3 mRNA can be detected in the adult working myocardium. Transfection of Zfhx3 strongly reduced peak INa density (from −75.0±6.6 to −30.9±2.9 pA/pF; n≥26, P<0.001). In contrast, Zfhx3 silencing augmented INa density compared to cells transfected with scrambled siRNA (from −65.9±8.9 to −104.6±10.8 pA/pF; n≥8, P<0.05). Neither Zfhx3 expression nor silencing modified time and voltage dependence of activation and inactivation or the reactivation kinetics. Zfhx3 significantly reduced transcriptional activity of human SCN5A, PITX2 and TBX5 minimal promoters and, consequently, the mRNA and protein expression levels of Nav1.5, Pitx2c, and Tbx5 were diminished (n≥6, P<0.05). In cells transfected with Zfhx3 together with Pitx2c, but not with Tbx5, INa density was significantly smaller than in cells expressing WT Zfhx3 alone (n≥15, P<0.05). Further WB experiments demonstrated that Zfhx3 increased the expression of Nedd4–2 ubiquitin-protein ligase, which ubiquitinates Nav1.5 channels and favors their proteasomal degradation. Conclusions Zfhx3 inhibits INa as a result of a direct repressor effect on the SCN5A promoter, the modulation of Tbx5-increasing and Pitx2-decreasing effects on the INa, and the enhancement of Nav1.5 channel degradation. We propose a novel and complex mechanism that regulates the expression of sodium channels and the density of the INa, which are critical for the control of cardiac excitability. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Economía y CompetitividadComunidad Autόnoma de MadridCellular transfections provide powerful experimental tools to study gene regulation. However, endothelial cells are difficult to transfect. Therefore, the present studies were performed to optimize transfection techniques in human endothelial cells. To study transfection rates, endothelial cells were transfected with the pGL3 vector, containing the luciferase reporter gene, complexed with several currently available liposomes, such as different Perfect Lipid (pFx) mixtures, DMRIE‐C, or lipofectin. The optimal transfection rate was achieved in cells transfected for 1.5 h with 5 mg/mL of DNA plasmid in the presence of 36 mg/mL of pFx‐7. Transfections mediated by other liposomes were less efficient. Usefulness of the optimized transfection technique was confirmed in cells transfected with NF‐kB or AP‐1‐responsive constructs. We conclude that among several currently available liposomes, pFx‐7 appears to be the most suitable for transfections of cultured human endothelial cells. Acknowledgements: Supported by NIH; NS39254, MH63022, and AA013843.
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The study aimed at improving gene transfection efficiency. We used PEI-mediated plasmid pEGFP-N1 to transfect NIH-3T3 cells in this study. NIH-3T3 Cells were divided into two groups to conduct one and two transfection,and observed under fluorescent inverted microscope at 48 h after transfection. The results showed that some cells emited green fluorescence,and green fluorescent cells in two transfection group were more than one transfection group. Calculated transfection efficiency to showed that two transfection method could significantly improve gene transfection efficiency.
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OBJECTIVE To study GDFN gene to transfect the OECs came from the surrounding tissue,analyze the expression level of transfected cell,and to provide the basis for the gene therapy for SCI.MOTHODS We extracted the OECs came from the surrounding tissue,used recombination adenovirus as the vector,transfected the GDFN to the OECs,and analyzed the expression level of the transfected cell by using bio-medical method.RESULTS GDFN was transfected into OECs,the transfection efficiency was 77%,the expression level of transfected cell was 30.3 ng/106/24h,and the expression level was steady.CONCLUSION Gene therapy for SCI by using GDFN as target gene to transfect OECs was feasible.
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Objective: To evaluate the transfection efficiency and optimal conditions of siRNA.Methods:Synthysized siRNA were transfected into human umbilical vein endothelial cells,we identified the rate of the transfection by fluorescent microscope and Western blotting.Results:The transfection rate of 35% was attained when FITC labeled negative siRNA was transfected into cells with siPORT~TM NeoFX~TM.Conclusion:The transfection activity of siRNA is observed,which provides a basis for finding the better method to check the rate of the transfection.
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OBJECTIVE:To explore the transfection effects and cell toxicity of mock miRNA at different concen-trations,liposome at different concentrations and different transfection times in human bronchial epithelial cells in vitro and optimize transfection conditions.METHODS:16-HBE cells were incubated with 0,10,30 and 50 nmol/L Cy3 labelled mock miRNA and 0,0.1%,0.3% and 0.5% liposome.The transfection effects in the cells were examined by fluorescence microscopy and were evaluated by fluorescence intensity analysis after 6,12 and 24 h of transfection.Cell activity was measured after 24 h transfection with different transfection conditions by WST-8 method.RESULTS:Intracellular fluorescence intensity was enhanced with increasing mock miRNA and liposome concentration and transfection time.The transfection effects of 30 and 50 nmol/L mock miRNA transfection groups was significantly higher than 10 nmol/L mock miRNA transfection group(P0.05).The transfection effects of 0.3% and 0.5% liposome groups were obviously higher than 0.1% liposome groups(P0.05).Intracellular fluorescence intensity after 24 h transfection was remarkably increased those that after 6 and 12 h transfections(P0.05).There was no significant difference in cell toxicity between transfection groups and control group(P0.05).CONCLUSION:16-HBE cell could be used in the research of liposome-mediated delivery of mock miRNA.Mock miRNA and liposome concentration and transfection time are important influence factors of transfection.Optimal transfection effects could be obtained after 24 h transfection with 30 nmol/L mock miRNA and 0.3% liposome.There was no effect on the cellactivity after transfection.
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Objective To compare the advantages and disadvantages of three different transfection reagents for mediated transfection of siRNA to mosquito cells and choose the most suitable for cell viability assay after transfection.Methods The specific siRNA was transfected into mosquito cells using FuGENEHD,X-tremeGENE,and siPORT Amine transfection reagents.Quantitative Real-Time PCR was performed to access gene expression 48 hr after transfection.The transfection efficiency,cytotoxicity,and use of different reagents were compared.Results Transfection efficiency was 70% for FuGENEHD,85% for X-tremeGENE,and 29% for siPORT Amine.FuGENEHD and siPORT Amine had lower cytotoxicity than did X-tremeGENE.During transfection,FuGENEHD in the culture medium had to be replaced once,X-tremeGENE had to be replaced twice,and siPORT did not have to be replaced.Conclusion The efficiency of transfection reagents significantly affected results.FuGENEHD has moderate transfection efficiency,low cytotoxicity,and was only replaced once during transfection.X-tremeGENE has high transfection efficiency but high cytotoxicity and was replaced twice during transfection.siPORTTM Amine has low cytotoxicity and was not replaced but has low transfection efficiency.
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We have studied different methods for efficient DNA transfection in neuroblastoma cell line. Here we report that the optimum transfection mode by nucleofection method yielded the maximum efficiency for SK-N-SH cells. To optimize transfection we selected different methods like electroporation, liposome mediated and PEI transfection. The DNA that was transfected was pmaxGFP plasmid (3.49kb). Our results suggest that the most efficient method for DNA transfection is very important to study gene expression and regulation by finding ways to deliver DNA into the difficult to transfect mammalian cells. The transfection efficiency by electroporation was ~60-70%, Lipofection was ~ 18-20% and PEI yielded <1%.
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