Expression of HCN4 protein and If in cardiac fibroblasts of rats transfected with human TBX1 8 gene and its effect on cardiomyocyte pulsation frequency
QUAN Dajun
CAO Zheng
YANG Mei
LI Sha
Abstract：Objective To observe the expression of HCN4 protein,If and its effect on the pulsation frequency of car-diomyocytes in cardiac fibroblasts transfected with human TBX18 gene.Methods The hearts were extracted after decapi-tation of the newborn SD rats,and the fibroblasts and cardiomyocytes were isolated and cultured.Adenovirus vector Ad-GFP-TBX18 was constructed and the virus titer was purified to 1 ×1010 TU/mL.The fibroblasts were divided into groups A,B and C at the time of passage 2 to 5.After 24 hours of culture,groups A and B were transfected with Ad-GFP-TBX18 and pHBAd-MCMV- GFP,respectively,and group C without any treatment.The mRNA expression of TBX18 mRNA was detected by RT-PCR.The hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4)protein was detec-ted by Western blotting.The hyperpolarization activated inward current (If)was recorded by voltage clamp.The fibroblasts of each group were co-cultured with neonatal rat cardiomyocytes to observe the frequency of cardiomyocytes in neonatal rats. Results The relative expression levels of TBX18 mRNA in the groups A,B and C were 98.32 ±6.21,1.03 ±0.05 and 1.02 ±0.02,respectively.The relative expression of TBX18 mRNA in the group A was higher than that in the groups B and C (all P<0.05).There was no significant difference in the relative expression of TBX18 mRNA between group B and group C.The relative expression of HCN4 protein in the groups A,B and C were 0.49 ±1.21,0.12 ±0.02,and 0.09 ±0.01,respectively.The relative expression of HCN4 protein in the group A was higher than that in the groups B and C (P<0.05).There was no significant difference in the relative expression of HCN4 protein between group B and group C. About 30% of the cells in group A were able to detect If,whereas in the groups B and C we did not detect If.At the same time,the frequency of cardiomyocyte pulsation in group A was higher than that in the groups B and C (all P<0.05 ). There was no significant difference in the frequency of cardiomyocytes between groups B and C.Conclusion HCN4 protein is highly expressed in rat cardiac fibroblasts transfected with human TBX18 gene and the If currents are present,which may promote the pulsation of neonatal rat cardiomyocytes.
Keywords：TBX18 genegene transfectionfibroblastcardiomyocytehyper-activated cyclic nucleotide-gated cation channel 4hyperpolarization-activated inward currentheart pacinganimal experiment
Publication Date：2016-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 21-24 )