Vascular Smooth Muscle Cell Phenotypic Transition Regulates Gap Junctions Of Cardiomyocyte

Objective:Our study aimed to investigate the effect of synthetic vascular smooth muscle cells（SMCs）on gap junction proteins in cardiomyocytes.Methods:1.Extraction of vascular smooth cells（SMC）from the pulmonary veins of Norway rats.TGF-β₁（1ng/ml for 24 hours）was used to induce the vascular SMCs switching to the synthetic phenotype.RT-PCR was used to detect the expression of collagen I,vimentin,calponin and micro RNA 27b.2.Established direct contact and non-contact co culture systems of SMCs and HL-1 cells and divided into three groups:HL-1 cells（HL-1 group）,HL-1 cells co-cultured with contractile SMCs（HL-1/SMC group）,HL-1 cells co-cultured with synthetic SMCs（HL-1/SMCTGFβ1 group）.18-α-GA（50μM for 24 hours）was used to inhibit gap junctions of SMCs.Western blotting was used to detect the expression of Cx43,Cx40 and Cx45 in HL-1 cells,and RT-PCR to test micro RNA 27b and ZFHX3 in vascular SMCs or in HL-1 cells;3.Using the condition media of contractile SMCs and synthetic SMCs cultured HL-1 cells.Western blotting was used to detect the expression of Cx43,Cx40 and Cx45 in HL-1 cells,and RT-PCR to test micro RNA 27b in vascular in HL-1 cells.4.Lucifer yellow dye was used to stain contractile SMCs and synthetic SMCs.Then HL-1 cells were co-cultured with stained contractile SMCs or synthetic SMCs.The fluorescence intensity of Lucifer yellow biocytin migrated to HL-1 cells was detected.Results:1.Expression ofα-SMA was positive in SMC extracted from the pulmonary veins of Norway rats;2.After SMCs were treated with TGF-β₁,the expression of collagen I and vimentin was increased while the expression of calponin was decreased.（p<0.05）;3.In the direct transmembrane co-culture system,Cx43 was significantly increased,and Cx40 and Cx45 were decreased in HL-1 cocultured with synthetic SMCs（p<0.05）;4.In non-contact co-culture system,the expression of Cx43,Cx45 and Cx40 was comparable in three groups;5.In condition media culture model,the expression of Cx43,Cx45 and Cx40 was comparable in three groups;6.The fluorescence intensity of Lucifer yellow was higher in HL-1 cocultured with synthetic SMCs than that in the cells cocultured with contractile SMCs,which was reversed by18-α-GA administrated to SMC in advance;7.The expression of micro RNA 27b was increased in HL-1 cocultured with synthetic SMCs,which was attenuated markedly by 18-α-GA administrated to SMC in advance.8.The expression of ZFHX3 was decreased in HL-1 cocultured with synthetic SMCs,which was reversed by 18-α-GA administrated to SMC in advance.Conclusion:The gap junction proteins of HL-1 were regulated by pulmonary venous SMCs undergoing phenotypic transition in this study,accompanied with the up-regulation of micro RNA27b and the down-regulation of ZFHX3 in HL-1 cells,which was associated with functional heterocellular gap junctions between HL-1 and pulmonary venous SMCs.