TBX18 Induced Cardiac Fibroblasts Transform Into Pacemaker-like Cells

Objective:To investigate the effect of fibroblasts after TBX18 gene transfection on the biological pacing of cardiomyocytes:1.To investigate the reprogramming effect of transcription factor TBX18 on cardiac fibroblasts in neonatal rats;2.To investigate the effect of fibroblasts after TBX18 gene transfection on the pulsation frequency of cardiomyocytes;3.To investigate the effects of fibroblasts on the pulsating frequency of cardiomyocytes after both transfecting TBX18 gene.Methods:1.Constructed recombinant adenovirus vector carrying human TBX18 gene(Ad-TBXl 8)and adenovirus vector with green fluorescent protein(Ad-GFP),then transfected into rat heart fibroblasts(CFs)and divided into experimental group A(CFs-TBX18),control group B(CFs-GFP)and blank control group C(CFs).The expression of HCN4 protein,Cox43 protein and Cox45 protein was detected in the cells cultured for 8 days.The expression of α-SA,MHC and cTn-Ⅰ protein was detected and the number of intracellular calcium sparks was recorded.Finally,the expression of a-SMA protein and vimentin protein were detected.2.CFs transfected by TBX18 were detected by patch bolus for observing the current of If.CFs-TBX18 were co-cultured with neonatal rat cardiomyocytes and divided into 4 groups:NRVM group,NRVM + CFs group,NRVM + GFP-CFs group with the NRVM + TBX18-CFs group.Each group was cultured respectively to 14 days and observed the pulse frequency.3.Both CFs and NRVM were transfected with TBX18 and divided into 5 groups:NRVM group,TBX18-NRVM group,TBX18-NRVM CFs group,TBX18-NRVM GFP-CFs group and TBX18-NRVM group.Each group was cultured for 8 days and beating frequency observed between groups of cells.Results:1.CFX-TBX18 did not show morphological changes and no spontaneous pulsation was observed,and HCN4 protein was highly expressed,CX43 and CX45 protein expression was less;CFs-TBX18 low expressed cTn-Ⅰ protein and no calcium sparks were recorded in the cells;the expression of a-SMA in CFs-TBX18 was higher than that of other groups,and the Vimentin protein was observed in all cells.2.Voltage-dependent If current was recorded by patch-clamp in CFs-TBX18;at the 8th after co-culture of NRVM and fibroblasts,the beat frequency was observed higher in CFs-TBX18 group than NRVM,but lower in CFs-GFP and CFs group,there was a significant difference between the two groups(P<0.05).3.After a total of 7 days’ culture,we found TBX18 could significantly increase the beating frequency of NRVM(94.20±7.38 vs.62.10±11.67,P<0.001).Compared to TBX18-NRVM group,co-cultivation of GFP-CFs or CFs with TBX18-NRVM have no obvious changes on beating frequency,but CFs had tend to slow down the beating frequency.Co-cultivation of TBX18-CFs and TBX18-NRVM can significantly improve the beating rate in the co-cultured system(118.50±7.25 vs.82.80±15.82,P<0.001).Conclusions:After being transfected with TBX18,CFs was transformed into myofibroblasts(MCFs)with high expression of HCN4 protein and low of CX43 and 45 proteins,and could increase the frequency of NRVMs and TBX18-NRVMs in co-culture system.It is speculated that injection of TBX18 heart can not only reproduce the cardiomyocytes,fibroblasts can also exert biological pacing function.