| Objective :To explore the effect of mitochondrial MICU1 in the development of sepsis-acquired skeletal muscle weakness and its possible molecular mechanism.MethodsPart Ⅰ60 C57/BL 6J mice were purchased and 40 of them were selected to construct sepsis models using cecum ligation and puncture(CLP),and the mice were randomly divided into four groups:(1)Sham group(n=8);(2)CLP modeling 6 h group(CLP-6 h group,n=10);(3)CLP modeling 12 h group(CLP-12 h group,n=10);(4)CLP modeling 24 h group(CLP-24 h,n=12).At the corresponding modeling time points,grip strength and compound muscle action potential(CMAP)of mice were detected.The expression levels of inflammatory factors interleukin-6(IL-6)and tumor necrosis factor α(TNF-α)were detected with blood collected from the heart of mice.After the mice are anesthetized with pentobarbital sodium,the diameter,cross-sectional area(CSA)of tibial anterior(TA)muscle fibers were detected by H&E staining.Western blotting and q PCR methods were used to detect mitochondrial calcium uniporter complex(mt CU),which includes mitochondrial calcium uptake protein 1(MICU1),mitochondrial calcium uptake protein 2(MICU2),mitochondrial calcium uniporter(MCU)and essential MCU regulator(EMRE).In addition,muscle wasting related proteins: muscle ring-finger containing protein 1(Mu RF1)and muscle atrophy F-box protein(MAFbx)were also be detected.Another 20 SPF male mice were transfected with adenoassociated virus(AAV)by TA muscle injection.24 μL of AAV was transfected into the left TA muscle of each group as a control group,labeled as AAV-C group;the right TA muscle was transfected with the same dose of AAV carrying the MICU1 target gene to enhance MICU1 expression,labeled as AAV-M group.After 4 weeks,a sepsis model was constructed using CLP.Mice were randomly divided into 2 groups:(1)Sham group(AAV-C-Sham,AAV-M-Sham;n=8),and(2)CLP modeling 24 h group(AAV-C-C-CLP,AAV-M-CLP;n=12).Mice grip strength and CMAP,muscle fiber diameter and expression levels of CSA and MICU1,Mu RF1,MAFbx were detected.Part ⅡC2C12 cells differentiated for 5 ~ 6 days were constructed with LPS(5 μg/m L)as a cellular sepsis model and randomly divided into four groups:(1)Control group;(2)LPS modeling 6 h group(LPS-6 h group);(3)LPS modeling 12 h group(LPS-12 h group);(4)LPS modeling 24 h group(LPS-24 h group).WB and q PCR methods were used to detect the expression level of Mu RF1,MAFbx and mt CU(MICU1,MICU2,MCU,EMRE).The calcium dye(Fluo-4 AM)was used to detect cytosolic calcium(c Ca2+)ion.Then,C2C12 cells were transfected with lentivirus carrying the MICU1 target gene to enhance MICU1 expression and labeled as LV-M group;empty plasmid was used as a negative control and labeled as LV-C group.The experiments were divided into two groups:(1)Control group(LV-C-Control,LV-M-Control)and LPS modeling 24 h group(LVC-LPS,LV-M-LPS).The expression levels of MICU1 and Mu RF1,MAFbx were detected by WB and q PCR,and cytoplasmic calcium ions were detected by the calcium dye Fluo-4 AM.Results:Part Ⅰ(1)Compared with sham group,the CMAP peak decreased,the duration and latency prolonged(P<0.05).Compared to the AAV-C-Sham group,there was no statistical difference in the AAV-M-Sham group(P>0.05),while AAV-C-CLP group had amplitude decreased,and duration and latency increased(P<0.05).Compared to the AAV-C-CLP group,the mice in the AAV-M-CLP group had higher amplitude,and duration and latency were shorter(P<0.05).(2)Compared with sham group,the diameter and CSA of muscle fibers decreased in the CLP-6 h,CLP-12 h and CLP-24 h groups(P<0.05).After the AAV injection,compared with the AAV-C-Sham group,there was no statistical difference in AAV-M-Sham group(P>0.05),while the diameter and CSA of muscle fibers in AAV-C-CLP group and the CSA in AAV-M-CLP group decreased(P<0.05);compared to the AAV-C-CLP group,the diameter and CSA of fibers in AAV-M-CLP group increased(P<0.05).(3)Compared with sham group,the expression level of Mu RF1,MAFbx protein and gene were increased in the CLP-12 h group and CLP-24 h group(P<0.05).After AAV intervention,compared with AAV-CSham group,there was no statistical difference in AAV-M-Sham group(P>0.05),and Mu RF1 and MAFbx protein and gene expression levels in AAV-C-CLP group and AAC-M-CLP group were increased(P<0.05);the protein and gene expression levels of Mu RF1 and MAFbx were significantly reduced in the AAV-M-CLP group compared with the AAVC-CLP group(P<0.05).(4)Compared to sham group,the levels of mitochondrial MICU1 and MICU2 proteins decreased in the CLP-24 h group(P<0.05),and the expression levels of MCU proteins in the CLP-6 h and CLP-12 h group were increased,as well as CLP-24 h group(P<0.05);the expression level of MICU1 and MICU2 protein in the CLP-24 h group decreased(P<0.05),and MCU protein in the CLP-12 h and CLP-24 h group increased compared to the sham group at the gene expression level(P<0.05).At the levels of protein and gene,there was no statistical difference in the expression of mitochondrial EMRE(P>0.05).(5)Compared with sham group,the expression level of muscle MICU1/MCU protein in the CLP-6 h,CLP-12 h,as well as CLP-24 h groups decreased(P<0.05),and the MICU1/MCU gene expression level in the CLP-24 h group decreased(P<0.05).Part Ⅱ(1)The expression level of Mu RF1 and MAFbx in three time points with LPS interventions were increased compared with control group(P<0.05).The expression of Mu RF1 at LPS-12 h and LPS-24 h and MAFbx in LPS-24 h increased compared with control group at the level of gene transcription.With lentiviral transfection,there was no statistical difference in LV-M-Control group(P>0.05),while the expression levels of Mu RF1 and MAFbx in LV-C-LPS and LV-M-LPS group increased compared with the LV-C-Control group(P<0.05).The expression of Mu RF1 and MAFbx in the LV-M-LPS group were increased compared with the LV-C-LPS group(P<0.05).There was no statistical difference in LV-M-Control group(P>0.05),while the expression levels of Mu RF1 and MAFbx in LV-C-LPS group increased compared with the LV-C-Control group at the level of gene transcription(P<0.05).In addition,the gene expression levels of the Mu RF1 and MAFbx in LV-M-LPS group were decreased compared with LV-C-LPS group(P<0.05).(2)The level of mitochondrial MICU1 and MICU2 proteins in the LPS-12 h and LPS-24 h group decreased(P<0.05),while MCU proteins increased in the LPS-24 h group compared with control group(P<0.05).Meanwhile,the MICU1 gene expression in LPS-12 h and LPS-24 h group and MICU2 gene expression in LPS-24 h group were decreased(P<0.05),and MCU gene expression in LPS-24 h group increased compared with control group(P<0.05).At the levels of protein and gene,there was no statistical difference in the expression of mitochondrial EMRE(P>0.05).(3)The expression level of MICU1/MCU protein at all time points with LPS intervention were decreased compared with control group(P<0.05).The expression level of MICU1/MCU in LPS-24 h group decreased compared with control group at the gene expression level(P<0.05).(4)When 2.5 μM calcium ions were added,the mitochondrial calcium(m Ca2+)release rate in three time points with LPS intervention increased compared with the control group(P<0.05).When 20 μM calcium ion were added,compared with control group,the m Ca2+ release rate of LPS-24 h group decreased(P<0.05).The expression level of Mu RF1 in C2C12 cells was correlated with m Ca2+ with LPS intervention,and the correlation coefficient R=0.8086.After lentiviral intervention,when 2.5 μM calcium ions were added,there was no statistical difference in LV-M-Control group compared with the LV-C-Control group(P>0.05),and the m Ca2+ release rate of cells in the LV-C-LPS group increased(P<0.05).The m Ca2+ release rate of LV-M-LPS group decreased compared with the LV-C-LPS group(P<0.05).When 20 μM calcium ions were added,there was no statistical difference in LV-M-Control group compared with the LV-C-Control group(P>0.05),while the m Ca2+ release rate in the LV-C-LPS group decreased(P<0.05).The m Ca2+ release rate of C2C12 cells in LV-M-LPS group increased compared with the LV-C-LPS group(P<0.05).Conclusion:(1)Sepsis causes mitochondrial calcium overload in the resting state and leads to skeletal muscle dysfunction by decreasing MICU1/MCU expression levels in mice and C2C12 cells;(2)LPS causes m Ca2+ overload in C2C12 cells at rest.When the c Ca2+exceeds the m Ca2+ uptake threshold,MICU1 loses the synergistic activation effect with MICU2,resulting in m Ca2+ uptake disorders;(3)Increasing the expression of MICU1 exogenously would improve sepsis-induced skeletal muscle dysfunction and muscle fiber atrophy ultimately by regulating disorders of m Ca2+ uptake. |
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