| Background Myocardial mitochondrial dysfunction is one of the important intrinsic characteristics of ischemic heart disease.Our previous study found that the protein expression level of mitochondrial ubiquitin ligase 1(Mul1)is significantly increased in ischemic myocardium,but its regulation in ischemic cardiomyocytes is still unclear.Objective To observe the regulatory effect of Mul1 on hypoxic cardiomyocytes and explore its mechanism.Methods 1.To investigate the effects of Mul1 on the growth viability and mitochondrial function of hypoxic cardiomyocytes:(1)Establishment and identification of hypoxic cardiomyocytes model: Rat cardiomyocytes H9C2 were cultured in a humidified incubator(5%CO2,94% N2,1% O2)for 12 hours.The morphology of cardiomyocytes was observed by inverted microscope,and the ultrastructure of cardiomyocytes was observed by transmission electron microscope.The cell viability was detected by CCK-8 method,and the level of lactate dehydrogenase(LDH)was detected by enzyme-labeled method.(2)To detect the expression of Mul1 in hypoxic cells: RT-q PCR and Western blot were used to detect the expression levels of Mul1 m RNA and protein in H9C2 cells.(3)Construction of Mul1 expression vector,experimental grouping and intervention: the lentiviral vector of Mul1 gene overexpression and knockdown in rats and the corresponding empty vector were constructed.Each lentiviral vector was transfected into H9C2 cells,and the cell lines with stable expression of Mul1 were selected by puromycin.The cells were divided into negative control(NC)group,overexpression empty vector control group,Mul1 overexpression group,knockdown empty vector control group,and Mul1 knockdown group,which were cultured in routine culture(5% CO2,21% O2)and hypoxia culture(5% CO2,94% N2,1% O2),respectively.(4)To examine the effects of Mul1 on the growth viability and mitochondrial function of hypoxic H9C2 cells: The changes of cell morphology were observed by inverted microscope,the ultrastructure of cardiomyocytes was observed by transmission electron microscope,the cell survival rate was dynamically observed by CBM intelligent cell detection platform,the cell viability was detected by CCK-8 method,and the content of LDH was detected by enzyme-labeled method.The level of mitochondrial membrane potential was detected by TMRE fluorescent probe method,the content of reactive oxygen species(ROS)was detected by fluorescence labeling method,and the concentration of adenosine triphosphate(ATP)was detected by chemiluminescence method.2.To explore the mechanism of Mul1 regulating mitochondrial function in hypoxic cardiomyocytes:(1)Identification of mitochondrial respiratory proteins that interact with Mul1 under hypoxia: Firstly,the mitochondrial respiratory proteins(target proteins)that interacted with Mul1 were screened by protein immunoprecipitation and mass spectrometry,and the target proteins that interacted with Mul1 were further predicted by Gene MANIA database and verified by protein immunoprecipitation.The co-localization of interacting proteins was observed by cellular immunofluorescence.(2)To detect the ubiquitination modification effect of Mul1 on target protein: I,H9C2 cells were transfected with Mul1 overexpression,Mul1 knockdown and corresponding empty vector,respectively,and then cultured in routine culture and hypoxia culture.Mitochondrial proteins were extracted and the changes in target protein levels were detected by Western blot.II,the half-life assay(cycloheximide inhibition assay)was used to detect the levels of target proteins in mitochondria of cells in each group at 0h,3h,and 6h of hypoxia.III,Target protein ubiquitination: Hypoxic cardiomyocytes were treated with proteasome inhibitor(MG132),and the target protein antibody was used for immunoprecipitation,and the ubiquitination level of the target protein was further detected by Western blot.Result Mul1 aggravated the hypoxic injury of cardiomyocytes:(1)The model of hypoxic cardiomyocytes was successfully established: compared with the normoxia group,H9C2 cells shrank and some cells fell off at 12 h of hypoxia.Under electron microscope,the mitochondrial structure of H9C2 cells in the normoxia group did not show abnormal changes.At 12 h of hypoxia,some mitochondrial outer membrane of H9C2 cells were dissolved,cristae was broken,and focal vacuolization was observed.Compared with the normoxia group,the cell viability of H9C2 cells decreased(P < 0.05)and the intracellular LDH content increased(P <0.05)after 12 h of hypoxia.(2)The expression levels of Mul1 m RNA and protein in H9C2 cells cultured in hypoxia for 12 h were higher than those in conventional culture group(P all <0.05).(3)Detection of interference efficiency of Mul1 lentiviral expression vector: After each lentiviral vector was transfected into H9C2 cells,the positive rate of green fluorescent protein expression was more than 70%.RT-q PCR and Western blot showed that the expression levels of Mul1 m RNA and protein in the Mul1 overexpression group were higher than those in the negative control group and the over-expression empty vector control group(P all < 0.05).The expression levels of Mul1 m RNA and protein in Mul1 knockdown group were lower than those in negative control group and knockdown empty vector control group(P all < 0.05).This result indicated that the interference efficiency of the constructed expression vectors was significant.(4)The effects of Mul1 on the growth vigor and mitochondrial function of hypoxic cardiomyocytes were examined by inverted microscope.After hypoxic culture,compared with the negative control group and the overexpression of empty vector group,the cytoplasm of Mul1 overexpression group showed coarse granule-like substances,and a large number of cells shrank and fell off.Compared with the negative control group and the knockdown empty vector group,most of the cells in the Mul1 knockdown group had better cell morphology and strong light transmission,and occasionally dead cells were observed.The results of transmission electron microscopy showed that compared with the control group,most of the cells in the Mul1 overexpression group had broken or dissolved mitochondrial cristal,and the cytoplasmic contents were significantly reduced.The mitochondrial structure of most cells in Mul1 knockdown group was normal,and occasionally cristae fracture was observed.The results of cell viability assay showed that the cell viability and cell survival rate in the Mul1 overexpression group were significantly lower than those in the negative control group and the overexpression empty vector control group after hypoxia culture(P all <0.05),and the cell viability and cell survival rate in the Mul1 knockdown group were higher than those in the negative control group and the knockdown empty vector control group(P all <0.05).The results of mitochondrial respiratory function test showed that the level of mitochondrial membrane potential and ATP in the Mul1 overexpression group were lower than those in the negative control group and the overexpression empty vector control group(P all < 0.05),and the content of ROS was increased(P all < 0.05).The levels of mitochondrial membrane potential and ATP in Mul1 knockdown group were higher than those in negative control group and knockdown empty vector control group(P all < 0.05),while the content of ROS was lower(P all < 0.05).2.Mechanism of Mul1 regulating hypoxic cardiomyocytes:(1)Protein immunoprecipitation-mass spectrometry analysis showed that ubiquinol cytochrome c reductase core protein I,ubiquinol cytochrome c reductase core protein I,ubiquinol cytochrome C reductase core protein UQCRC1)was one of the mitochondrial respiratory chain proteins that interacted with Mul1,and Gene MANIA database also found that UQCRC1 interacted with Mul1.Co-immunoprecipitation assay further confirmed the interaction between Mul1 and UQCRC1.Immunofluorescence assay showed that Mul1 and UQCRC1 were both expressed in the cytoplasm and co-localized.(2)Detection of ubiquitination modification of UQCRC1 by Mul1: I.We found that under normoxia and hypoxia culture,the protein expression level of UQCRC1 in Mul1 knockdown group was higher than that in negative control group and knockdown empty vector control group(P all < 0.05).There was no significant difference in the protein expression level of UQCRC1 between the over-expression Mul1 group and the negative control group or the over-expression empty vector control group(P all > 0.05).II.The results of half-life assay showed that the protein level of UQCRC1 in Mul1 knockdown group was higher than that in knockdown empty vector control group after 3h and 6h of treatment with CHX(P all < 0.05).III.The intracellular ubiquitination assay found that the level of UQCRC1 ubiquitination could be suppressed in the Mul1 knockdown group relative to the empty vector control group.Conclusion Mul1 aggravates hypoxia-induced cardiomyocyte injury,which is related to the impairment of mitochondrial respiratory function. |
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