| Background Cardiac aging is defined as the heart structural and functional abnormality caused by age-associated adverse stimuli accumulation,manifesting as the marked rise of the aging phenotype ratio in the myocardium.Previous studies have indicated that mitochondrial dysfunction is the key event that drives myocardial senescence and is closely related to the abnormal accumulation of P53,known as an aging marker.We preliminarily found the decreased expression of Trim69 in the heart tissue of aging mice,an E3 ubiquitination enzyme that has been reported to regulate the P53 level through the ubiquitination degradation pathway negatively.However,the effect of Trim69 on cardiac aging and aging-associated myocardial mitochondrial dysfunction remains unclear.Objective This study was conducted to explore the effect of Trim69 overexpression on cardiac aging and aging-associated mitochondrial dysfunction in a D-gal-induced aging model by transfecting lentivirus vectors with Trim69 into rat heart tissue and H9C2 cell line,to provide the theoretical basis for the therapeutic strategy of cardiac aging based on Trim69.Methods In vivo: The drug-induced senile animal model was established through subcutaneous injection of low-dose D-galactose in the nape of male SD rats.Experimental animals were randomly divided into the Sham group,D-gal group,D-gal+LV-NC group,and D-gal+LV-Trim69 group.The rats in the D-gal+LV-NC group and D-gal+LV-Trim69 group were transfected with recombinant lentivirus vector LV-NC and LV-Trim69 into rat myocardial tissue by myocardial multipoint injection after 4 weeks of drug induction respectively,while the D-gal group and Sham group were injected with the same quantity of isotonic saline.Efficiency of lentivirus was detected by fluorescence microscope.An ultrasonic probe special for animals was used to record the heart function of rats and evaluate the achievement of the aging model.The β-galactosidase staining was used to assess the cardiac aging of rats.Western blot and immunohistochemistry were used to determine the expression and cellular distribution of aging markers.The Masson staining,WGA staining,and immunohistochemistry of 3-NT were used to evaluate the aging-related cardiac phenotypes.Transmission electron microscopy was used to observe mitophagy and morphology of mitochondria in aging myocardium.Western blot was used to detect the expression of Trim69 and mitophagy-related proteins.In vitro: The H9C2 cells were induced to construct a senescent cell model by D-galactose treatment.Recombinant lentivirus with different multiplicity of infection(MOI)was infected into H9C2 cardiomyocytes.The efficiency of infection and optimal MOI value were assessed by flow cytometry and fluorescence microscopy,respectively.The group of cell experiment is the same as that of animals.Similarly,β-galactosidase staining was also used to evaluate the aging phenotype of H9C2 cells.Mitochondrial membrane potential and mitophagy of H9C2 cells were detected by JC-1 staining and mitophagy detection kit,respectively.Western blot was used to detected the expression of Trim69,aging markers and mitophagy-related proteins.Co-IP and Western blot were used to detect the interaction between Trim69 and P53 and the level of P53 ubiquitination.Results In vivo:The green fluorescence could be found in cardiac apex in the virus groups under fluorescence microscope,but not found in Control and D-gal group.1)Overexpression of Trim69 improved D-gal-induced cardiac dysfunction: after 8 weeks of nape subcutaneous injection of D-galactose,the left ventricular ejection fraction(LVEF)and short axis shortening rate(LVFS)of SD rats were significantly reduced.Overexpression of Trim69 markedly improved the LVEF and LVFS in D-gal-treated rats(P<0.05).2)Overexpression of Trim69 reversed the D-gal-induced cardiac aging: in D-gal group,the positive area of β-galactosidase staining was significantly expanded,which is accompanied by an increased level of aging marker P53 and P21,as well as the presence of aging-related cardiac phenotypes,including oxidative stress,cardiac hypertrophy,and fibrosis.Compared with D-gal+LV-NC group,it could be found that the proportion of aging staining positive areas in D-gal+LV-Trim69 was significantly reduced,meanwhile,the expression of P53,P21 and the aging-related cardiac phenotypes were also decreased(P<0.05).3)Overexpression of Trim69 activated mitophagy in D-gal-induced aging myocardial cells: the transmission electron microscope images showed that in the D-gal and D-gal+LV-NC groups,the myocardial cells in aging rats suffered extensive mitochondrial damage when mitophagy was repressed.The results of Western blot also indicated that the expression of Trim69 and mitophagy-related proteins PINK1 and Parkin decreased significantly,while the level of autophagy substrate P62 increased.Overexpression of Trim69 markedly upregulated the expression of PINK1 and Parkin,while the level of P62 was decreased,with the activation of mitophagy and reduction of damaged mitochondria.(P<0.05)In vitro: the infection efficiency reached 90% with no obvious cell death when the MOI value was 75.1)Overexpression of Trim69 mitigated the D-gal-induced cellular senescence in myocardial cells: compared with the Control group,the proportion of β-galactosidase staining positive cells was significantly increased in D-gal and D-gal+LV-NC groups,which was decreased by about 30% in the D-gal+LV-Trim69 group.The expression of P53 and P21 were also markedly up-regulated in D-gal-treated groups but down-regulated in the D-gal+LVTrim69 group(P<0.05).2)Overexpression of Trim69 reversed the decreased mitochondrial membrane potential in D-gal-treated myocardial cells: the mitochondrial membrane potential of myocardial cells in D-gal and D-gal+LV-NC groups was remarkably decreased when compared with Control group,which was partially reversed in the D-gal+LV-Trim69 group(P<0.05).3)Overexpression of Trim69 activated mitophagy in D-gal-treated myocardial cells:in D-gal-treated groups,it could be found that both the ratio of mitophagy positive cells and the intensity of mitophagy signal were decreased when compared with Control group,accompanied by the decreased expression of Trim69,PINK1 and Parkin and increased level of P62.Overexpression of Trim69 up-regulated the expression of PINK1 and Parkin,thereby activating the mitophagy in D-gal-treated myocardial cells.(P<0.05)4)Trim69 directly mediate the ubiquitination of P53 in myocardial cells: the results of Co-IP suggested Trim69 directly interact with P53.The detection of ubiquitination showed overexpression of Trim69 leads to an increased ubiquitination level of P53.Conclusion Overexpression of Trim69 could promote ubiquitin degradation of P53,which further inhibits the abnormal accumulation of P53,therefore alleviate the cardiac aging and mitochondrial dysfunction. |
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