The Reprogramming Effect Of The Transcription Factor Tbx18 On The Ascending Aortic Smooth Muscles Cells

Objective:The study explored whether ascending aortic smooth muscle cells in vitro could be reprogrammed into pacemaker-like cells with human Tbx18.After overexpressing Tbx18 into vascular smooth muscle cells(VSMCs)of ascending aorta,we could observe the change of reprogrammed cells about the function and phenotype,and explore the effect of Tbx18 on reprogramming of ascending aortic smooth muscle cells in vitroMethods:Ascending aortic smooth muscle cells were cultured using tissue-block adherence.Cell morphology was observed under a light microscope after 4 to 7 days.VSMCs were randomly divided into Null group(blank group),GFP group(control group),Tbx18 group.The cells were transfected with Ad-Tbx18 in Tbx18 group,and the cells with the same amount of Ad-GFP in GFP group.After 4 days of transfection,the expressions of these specific genes NKx2.5,ISL1,Shox2,Tbx3,HCN4,Cx43 and c Tn I were detected by RT-q PCR and Western blot in three groups,as well the expression of HCN4 protein in Tbx18 and GFP groups by immunofluorescence.Then the transfected VSMCs at 4 day were cocultured with neonatal rat ventricular cardiomyocytes(NRVMs)for 5 days in vitro.Following that observe the change of beating rate in different groups,and detect the APs and inward current of the reprogrammed cells by patch clamp technology.Results:The cultured primary ascending aortic smooth muscle cells were long spindle-shaped and grew faster.After the treatment,the results showed that compared with the GFP group and the blank group,the expression of ISL1,Shox2,Tbx3,HCN4 and c Tn I m RNA and protein in the Tbx18 group were significantly up-regulated,the m RNA of Cx43 down-regulated,and the expression of Cx43 and NKx2.5 protein significantly decreased(P<0.05).HCN4 protein in Tbx18 group showed red fluorescence under the inverted fluorescence microscope,and almost coincided with the green fluorescence of the cell transfection and the blue fluorescence of the nucleus by immunofluorescence.After 3 days of co-cultivation,the Tbx18+VSMCs group formed a significant coupling with NRVMs,which the beating rate was significantly faster(178.00±7.55 bpm,P<0.05).And I_f current and the SAN-like AP could be detected by patch clamp.Conclusion:Tbx18 can significantly change the morphology and function of VSMCs.It indicated that Ad-Tbx18 has the potential to reprogram ascending aortic smooth muscle cells into pacemaker-like cells in vitro.