Expression Of HCN4 In Cardiac Fibroblasts,Cardiomyocytes And Endothelial Cells

BackgroundHyperpolarization activated nucleotides gating cationic ring channel 4(hyperpolarization-activated cyclic nucleotide gatedcation channels 4,HCN4)mainly expressed in heart sinoatrial node,has close relationship with pacemaker electric currents,therefore HCN4 gene is also known as cardiac pacemaker,HCN4 gene mutations can lead to sinus node dysfunction and abnormal function of cardiac conduction system development.There are few reports on whether HCN4 is expressed in cardiac fibroblasts,cardiomyocytes and endothelial cells after birth,and whether there are differences in expression at the cellular and tissue levels.This study attempted to investigate the expression of HCN4 in fibroblasts,cardiomyocytes and endothelial cells at the cellular and tissue levels.ObjectiveThe expression distribution of HCN4 in cardiac fibroblasts,cardiac myocytes and human umbilical vein endothelial cells,the expression pattern of HCN4 in in vitro fibroblasts,and the aging changes in newborn,adult and elderly SD rats were detected.Methods39 SD rats were divided into the newborn group(1-3 days),the adult group(2 months)and the elderly group(18 months).In addition,15 newborn SD rat hearts were digested by0.2% trypsin and 0.1% collagenase type II to obtain primary myocardial fibroblasts and primary myocardial cells.The expression of HCN4 in human umbilical vein endothelial cells,primary myocardial fibroblasts and primary myocardial cells was detected by cellular immunofluorescence and cellular immunohistochemistry.The expression of endothelial cells,fibroblasts and cardiomyocytes HCN4 in the hearts of newborn,adult and elderly SD rats was detected by tissue immunofluorescence and tissue immunohistochemistry.Theexpression of HCN4 in primary cardiac fibroblasts was detected by Western blotting technique and real-time fluorescence quantitative polymerase chain method,and the expression of HCN4 in SD rats of different age was detected by real-time fluorescence quantitative polymerase chain method.ResultsIn vitro cell culture showed that HCN4 was expressed in human umbilical vein endothelial cells,fibroblasts and primary cardiac myocytes.Cardiac muscle sections showed that HCN4 was expressed in fibroblasts and cardiac muscle cells in newborn,adult and elderly SD rats,but not in endothelial cells.Western blotting and PCR results of p1-p4 generation fibroblasts showed that the expression level of HCN4 gradually decreased with the generation of fibroblasts increasing,and the PCR results of left ventricle of rats at different months of age showed that the expression level of HCN4 gradually decreased with the increase of month of age,which was consistent with the expression level of cultured cells.conclusionThe expression of HCN4 decreased with the increase of age and cell generation.The expression of HCN4 was dominated by fibroblasts and some cardiomyocytes,while the expression of endothelial cells was uneven.