Mfn2-mediated Mitochondria-endoplasmic Reticulum Physical Coupling In The Role Of Copper-induced Autophagy In Duck Renal Tubular Epithelial Cells

In order to investigate the effect of high copper on mitochondria-endoplasmic reticulum physical coupling（mitochondria-endoplasmic reticulum physical coupling,MAM）of duck renal tubular epithelial cells and the role of Mfn2 mediated MAM in high copper-induced autophagy of duck renal tubular epithelial cells,duck renal tubular epithelial cells were cultured from 10-15 days old ducklings as experimental materials,and exposed to 0,100 and 200μM anhydrous copper sulfate（Cu SO₄）for 12 h.The morphology of MAM was observed,and mitochondrial calcium concentration and colocalization of IP3R and VDAC1 were determined.In addition,MAM related genes m RNA levels and protein levels were measured.Results showed that the distance between mitochondria and endoplasmic reticulum was longer,the number of MAM was decreased,and the cristae of mitochondria became vague and vacuolated,the interaction between MAM structural protein IP3R and VDAC1 was weakened,mitochondrial calcium level decreased,the m RNA expression levels of Mfn2,PACS2,IP3R and MCU were upregulated,the m RNA expression and protein level of Grp75 were upregulated;the protein expression levels of Mfn2 and VDAC1 were reduced in duck renal tubular epithelial cells incubated with Cu²⁺.In conclusion,high copper can cause structural and functional disorder of MAM in duck renal tubular epithelial cells.In addition,to further study the role of Mfn2 mediated MAM in copper-induced autophagy of duck renal tubular epithelial cells,the lentiviral over expression vector（Lv-Mfn2）and empty vector（Vec）of Mfn2 with multiplicity of infection（MOI）of 20were transfected into the cells,and the cells were divided into six groups:Control group（control group）,Cu group(200μM Cu²⁺)and Cu+Lv-Mfn2 group(200μM Cu²⁺+Lv-Mfn2 MOI=20),Lv-Mfn2 group（Lv-mfn2 MOI=20）,Cu+Vec group(200μM Cu²⁺+Vec MOI=20)and Vec group（Vec MOI=20）.The transmission electron microscopy was used to observe and tphotograph MAM and autophagosome morphological changes.The acidic autophagic vesicles and LC3 aggregation points were observed,the cell survival rate was determined,the mitochondrial calcium concentration and reactive oxygen species（ROS）level were measured by flow cytometry,the colocalization of IP3R and VDAC1 was analyzed,and the mitochondrial endoplasmic reticulum structure coupling and autophagy related genes were detected.The results showed that compared with Cu group,the distance between mitochondria and endoplasmic reticulum in Cu+Lv-Mfn2 group was shorter,the number of MAM was increased,the interaction between MAM structural protein IP3R and VDAC1 was increased,the mitochondrial calcium level and ROS level were upregulated,the m RNA expression levels of Mfn2,VDAC1 and IP3R were increased,and the protein expression levels of Mfn2 and VDAC1were increased,and the m RNA expression level of Grp75 was upregulated.At the same time,compared with Cu group,Cu+Lv-Mfn2 group had more severe cell injury,obviously reduced cell survival rate,increased autophagy,increased acidic autophagy vesicles and enhanced fluorescence intensity,increased LC3 immunofluorescence spots,upregulated m RNA expression levels of Atg5 and LC3B,reduced m RNA expression levels of P62,and reduced m RNA expression levels of Beclin1 and the protein levels of Atg14,LC3II/LC3I were upregulated.In conclusion,Mfn2 can aggravate copper induced autophagy by regulating the structure and function of MAM.