Role And Mechanism Of DDIT4 In The Regulation Of Cardiac Senescence

Part ? The expression of DDIT4 in senescent heart and the role for DDIT4 in the regulation of Dox-induced cardiac senescenceAims:The part aims to explore the expression level of DNA damage inducible transcript 4(DDIT4)in myocardial tissue of aged mice,and to examine the expression level of DDIT4in senescent myocardium induced by Doxorubicin(Dox).To test whether DDIT4overexpression or silencing would influence the development of cardiac senescence induced by Dox.Methods:(1)For age-induced senescence,C57BL/6 mice were divided into two groups:young(3?4 month)and old(22?24 month).Myocardial tissue samples were collected after sacrifice.The p16^INK4a and p21 positive cells were detected by immunofluorescence,and the protein expression levels of p16^INK4a and p21 were detected by Western blot to evaluate heart aging.Western blot was performed to determine the level of DDIT4 in myocardial tissue.(2)8-week-old wild-type male C57BL/6 mice were selected as subjects,and cardiac senescence was induced by intraperitoneal injection of Dox.The p16^INK4aand p21 positive cells were detected by immunofluorescence,and the protein expression levels of p16^INK4aand p21 were detected by Western blot to evaluate cardiac senescence.Western blot was performed to determine the level of DDIT4 in myocardial tissue.(3)Adeno-associated virus AAV9-NC,AAV9-DDIT4,AAV9-sh NC,AAV9-sh DDIT4were injected via the tail vein.Four weeks after virus injection,senescence was induced by intraperitoneal injection of Dox.The mice were divided into 4 groups:Ctrl,Dox,Dox+AAV9-DDIT4,Dox+AAV9-sh DDIT4.After 4 months,cardiac function was measured by echocardiography in the 4 groups.Cardiac tissue samples were collected after sacrifice.The expression level of DDIT4 was detected by immunofluorescence and Western blot to detect the efficiency of overexpression and silencing.HE staining was detected to assess heart dilatation.Masson staining and immunofluorescence staining of?SMA were detected to assess cardial fibrosis.Heart senescence was detected by immunofluorescence staining of p16^INK4a and p21,as well as the protein expression levels of p16^INK4a and p21 by Western blot.RT-PCR was used to determine m RNA levels of IL-1?,IL-6,IL-12 and TNF-?to evaluate SASP.Results:(1)Compared with young group mice,immunofluorescence showed increased?SMA positive cells in aged mice,as well as increased p16^INK4aand p21 positive cells.Western blot showed that the expression levels of p16^INK4a and p21 increased in aged mice.Simultaneously,the protein expression level of DDIT4 increased significantly.(2)Compared with normal control mice,immunofluorescence showed increased p16^INK4aand p21 positive cells in Dox-treated mice.Western blot showed that the expression levels of p16^INK4a and p21 increased in Dox-treated mice.Simultaneously,the protein expression level of DDIT4 increased significantly.(3)Immunofluorescence and Western blot analysis showed that AAV9-DDIT4significantly increased the protein expression level of DDIT4 in mouse heart,while AAV9-sh DDIT4 significantly silenced the protein expression level of DDIT4 in mouse heart.Mice in the Dox+AAV9-DDIT4 group exhibited higher levels of p16^INK4a and p21positive cells compared to the Dox+AAV9-NC group;whereas AAV9-sh DDIT4 infection reduced p16^INK4aand p21 positive cells.In addition,RT-PCR analysis indicated that DDIT4overexpression produced higher levels of SASP,while AAV9-sh DDIT4 reduced SASP levels.Furthermore,echocardiography showed that mice in the Dox+AAV9-DDIT4 group exhibited higher levels of LVEDd and LVEDs than the Dox+AAV9-NC group;whereas AAV9-sh DDIT4 significantly reduced LVEDd and LVEDs induced by Dox.In addition,interstitial fibrosis stimulated by Dox was amplified by DDIT4 overexpression,while silencing of DDIT4 expression reduced Dox-induced interstitial fibrosis.Conclusions:(1)The expression level of DDIT4 in aged heart was significantly upregulated.(2)The expression level of DDIT4 in Dox-treated heart was significantly upregulated.(3)DDIT4 overexpression promoted Dox-induced cardiac cell senescence,heart dilatation and fibrosis,while DDIT4 silencing attenuated Dox-induced cardiac cell senescence,heart dilatation and fibrosis.Part ? The role of DDIT4 on cardiomyocyte senescent at the cellular levelAims: This part was designed to test the expression level of DDIT4 in Dox-induced senescent cardiomyocytes,and the role of DDIT4 on cardiomyocyte senescent at the cellular level.Methods : Cultured H9c2 cardiomyocytes were stimulated with DMSO and Dox respectively to establish a cardiomyocyte senescence model,which were divided into Vehicle group and Dox group.Cardiomyocyte samples were collected for detection of DDIT4 expression levels.H9c2 cardiomyocytes were infected with recombinant lentiviral vectors Lv-DDIT4 and Lv-sh DDIT4 to overexpress or silence the expression of DDIT4,under the control of Lv-NC and Lv-sh NC.Cardiomyocytes infected with lentivirus were collected for SA-?-gal staining 48 hours after Dox stimulation.Western blotting was performed to detect the protein expression levels of p16INK4 a and p21.RT-PCR was used to determine m RNA levels of IL-1?,IL-6,IL-12 and TNF-? to evaluate SASP.Results: In cultured H9c2 cardiomyocytes,Dox induced the expression of DDIT4 in a time-dependent manner,peaked at 72 h,and paralleled with the increase of p16INK4 A and p21.Besides,Dox treatment significantly increased the level of SA-?-gal staining in H9c2 cardiomyocytes.Lv-DDIT4 pre-infected H9c2 resulted in a further increase in SA-?-gal staining,whereas Lv-sh DDIT4 pre-infected H9c2 partially eliminated the level of SA-?-gal staining.At the same time,we found that the expression levels of p16INK4 a and p21 were increased in Dox-treated H9c2 cardiomyocytes,which were further enhanced by DDIT4 overexpression,while DDIT4 silencing partially attenuated the high expression of p16INK4 a and p21.In addition,SASP levels were also elevated after Dox treatment of H9c2 cardiomyocytes,which were even higher in Lv-DDIT4-infected cardiomyocytes,while silencing DDIT4 reduced SASP levels.Conclusions: DDIT4 was increased in Dox-induced senescent cardiomyocytes.Overexpression of DDIT4 promoted Dox-induced premature senescence of cardiomyocytes,whereas silencing DDIT4 inhibits Dox-induced premature senescence of cardiomyocytes.Part ? The molecular mechanisms of DDIT4 in the regulation of Dox-induced cardiomyocyte senescenceAims: To investigate the molecular mechanisms of DDIT4 in the regulation of Dox-induced cardiomyocyte senescence.Methods : Cultured H9c2 cardiomyocytes were stimulated with DMSO and Dox respectively to establish a cardiomyocyte senescence model,which were divided into Vehicle group and Dox group.H9c2 cardiomyocytes were infected with recombinant lentiviral vectors Lv-DDIT4 and Lv-sh DDIT4 to overexpress or silence the expression of DDIT4,under the control of Lv-NC and Lv-sh NC.The phosphorylation and total protein levels of signaling pathway molecules in cardiomyocyte of each group were detected by Western blot.To determine the effect of p38 MAPK and NF-?B activation on the expression of DDIT4 induced by Dox,H9c2 cardiomyocytes were pretreated with p38 inhibitor SB203580 or NF-?B inhibitor BAY-11-7082,followed by Dox stimulation.Western blotting was performed to detect the protein expression level of DDIT4.In order to verify the effect of p38 MAPK on the regulation of cardiomyocyte senescence,H9c2 cardiomyocytes were preinfected with Lv-DDIT4,then treated with SB203580,followed by Dox stimulation.Western blot was used to detect the expression levels of p16INK4 a and p21 to evaluate cardiomyocyte senescence.RT-PCR was used to determine the m RNA levels of IL-1 ?,IL-6,IL-12 and TNF-? to evaluate SASP.Results: In cultured H9c2 cardiomyocytes,pretreatment with SB203580 partly abolished Dox-induced upregulation of DDIT4.However,BAY-11-7082 did not significantly inhibit Dox-induced DDIT4 expression.In addition,SB203580 significantly decreased the expression levels of p16INK4 a and p21 induced by Dox.However,Lv-DDIT4 pre-infected H9c2 partially blocked the effect of SB203580.NF-?B p65 in H9c2 cardiomyocytes was activated 0.5 hours after Dox treatment.Silencing DDIT4 reduced the phosphorylation level of NF-?B p65,while exogenous DDIT4 overexpression further activated NF-?B p65.Consistently,immunofluorescence analysis indicated that DDIT4 silencing inhibited Dox-induced NF-?B p65 nuclear translocation in H9c2 cardiomyocytes.Conclusions: p38 MAPK acted as an upstream mediator of DDIT4 in Dox-induced premature senescence of cardiomyocytes.NF-?B activation was required for DDIT4 to accelerate cardiomyocyte senescence.