The Mechanism Of Cardiac Fibrosis Mediated By Angiotensin ?-Role Of TRPM7 Channels

Background:Transient receptor potential melastatin 7(TRPM7),a bifunctional channel protein owning both cation permeability and kinase activity,plays an important role in the pathophysiological process of many cell types,such as vascular smooth muscle cells,human glioma cells and mouse cortical astrocytes,etc.However,whether TRPM7 channels play a key role in cardiac fibrosis through mediating the functional change of cardiac fibroblasts(CFs)induced by angiotensin II(Ang II)remains unknown.Objective:To investigate the role of TRPM7 ion channel proteins in the functional change of CFs and the development of CF induced by Ang II.Methods:CFs derived from SD rat were separated,primary and passage cultured using the method of enzyme-chemical digestion.The growth state and morphology during different periods of CFs were observed by optical microscope.Compare the transfection efficiency of adenovirus vector and lentivirus vector on CFs and explore the best multiplicity of infection(MOI)values of adenovirus vector on CFs.Build adenovirus vector carrying TRPM7-siRNA.The green fluorescent protein '(GFP)gene and adenovirus vector carrying TRPM7-siRNA were transfected into CFs.After 48 hours since virus transfection,CFs morphology and green fluorescence protein expression were observed under fluorescence microscope,RT-PCR and Western blot technologies were applied to detect the efficiency of TRPM7 gene silencing on CFs.First,1 umol/L Ang ? treat CFs to five different time points(0 h,6 h,12 h,24 h,48 h),Western blot technology was used to find TRPM7 protein expression level on CFs in aforementioned different time points,so as to explore the best time point of 1 umol/L Ang ? intervening CFs.Then,using 1 umol/L Ang II treat CFs transfeced with adenovirus vector carring GFP and TRPM7-siRNA respectively to 24 hours,whole-cell patch clamp method was applied to detect TRPM7 current amplitude on CFs membrane in each group,CCK-8 assay was used to detect the proliferation of CFs,immunofluorescence techonology was used to detect the differentiation of CFs,Western blot was applied to detect expression level of Ki67,PCNA,aSMA,Collagen ? and Collagen ? on CFs respectively,RT-PCR was used to detect the mRNA level of Collagen I and Collagen III on CFs.Results:We successfully completed the isolation,primary and passage culture of CFs in vitro.CFs were in good growth condition in vitro,with long spindle growth,cell bodies become flat along with the increased culture time,and extende pseudopodia.By comparison,we found that the transfection efficiency of adenovirus vector were obviously higher than lentiviral vector at the same MOI value,and the best MOI value of adenovirus vector transfecting CFs was 50(MOI=50).Afterwards,we successfully constructed the recombinant adenovirus vector pHBAd-U6-GFP-TRPM7-siRNA(Ad-TRPM7-siRNA);and Ad-GFP was seen as the control virus group.Transfecting CFs with adenovirus to 48 hours setting MOI as 50,RT-PCR and Western blot showed that TRPM7 expression level was obviously lower than that in the control virus group.Ang II stimulation caused time-dependent TRPM7 protein expression increase,TRPM7 current amplitude amplification,CFs proliferation and differentiation ability enhancement and collagen synthesis increase.However,downregualting the expression of TRPM7 on CFs with Ad-TRPM7-siRNA significantly suppressed the increased proliferation,differentiation and collagen synthesis of CFs induced by Ang ?.Conclusion:Our results identified TRPM7 channel as a pivotal member was associated with CFs functional change induced by Ang ?,and suggested that TRPM7 channel might represent a promising therapeutic strategy for the treatment of fibrosis related cardiac diseases.