Regulatory Role And Mechanism Of Stress-induced Cardiac Senescence In The Heart Remodeling

BackgroundDespite advances in the treatment of acute myocardial infarction(AMI),many patients still develop progressive heart failure in several years as a result of declining cardiac function.This situation suggests that there are missing pathophysiological mechanisms,which motivates researchers to explore viable strategies to preserve post-AMI cardiac function.Cellular senescence was traditionally defined as the state of irreversible cell cycle arrest after cells reached a maximal number of divisions;this process is termed as "replicative senescence".Functionally,it was believed that senescence caused senescence-related functional decline.However,these concepts have been redefined by recent studies.Stress-induced senescence is used to describe the phenomenon in which various stressors,such as oncogenic events and ionizing radiation,instigate cellular dysfunction.Mechanically,senescent cells can affect surrounding cells and play active roles in tissue remodeling by secreting such proteins as proinflammatory cytokines,chemokines,and growth factors(Senescence-associated secretory phenotype,SASP).Moreover,cardiomyocyte senescence is implicated in the myocardial damage caused by doxorubicin and obesity.However,the contribution of cardiomyocyte senescence to the heart remodeling has not been thoroughly elucidated to date.Mitochondrial aldehyde dehydrogenase(ALDH2)plays a role in the physiology and pathophysiology of a number of organ systems through by detoxification of exogenous and endogenous aldehydes into non-reactive acids.Compared with ROS,aldehydes(such as 4-hydroxy-2-nonenal,4-HNE and malondialdehyde,MDA)are enduring toxicant agents,forming an adduct with DNA,RNA and proteins.Lines of evidence suggest that chronic accumulation of aldehydes resulting from ALDH2 deficiency is responsible for age-related diseases.However,the interaction between ALDH2 and myocardial premature senescence remains unknownObjectives1.We tested whether cardiomyocytes underwent stress-induced senescence in response to inj ury.2.We aimed to study the role of cardiomyocyte senescence in the heart remodeling after AMI,and to investigate the underlying mechanism.3.We sought to study the interaction between ALDH2 and cardiomyocyte senescence after AMI.Methods1.Experimental AMI was produced by ligation of the left anterior descending(LAD)coronary artery.At 1st day,1st week,2nd week,4th-week post-AMI,senescence markers including p16INK4a,p21CIP1/WAF1,and SA-?-gal staining were analyzed in the hearts by immunostaining and Western blot.2.Stress-induced senescence is used to describe the phenomenon in which various stressors,such as oncogenic events and ionizing radiation,instigate cellular dysfunction.Upon DNA damage,GATA4 is activated and in turn,initiates the senescence-related pathways.In this study,we utilized y-H2AX immunostaining to assess the DNA damage in postinfarction hearts and the expression level of downstream GATA4.Different from apoptosis,senescent cells can affect surrounding cells and play active roles in tissue remodeling by secreting such proteins as proinflammatory cytokines,chemokines,and growth factors(senescence-associated secretory phenotype,SASP).GATA4 is required to maintain senescence and SASP.To examine the effects of GATA4 inhibition on activated SASP,we utilized adeno-associated virus 9(AAV9)-Gata4-shRNA to achieve GATA4 knockdown in vivo.Western blot was used to assess the changes of selected SASP factors including IL-la,TNF-?,MCP-1,and CCN1.Echocardiography was further used to measure heart functions,and Masson staining was performed to assess the fibrosis after GATA4 knockdown.3.Neonatal rat cardiac myocytes(NRCMs)were isolated as a published standard protocol.The senescent cardiomyocyte model was established by culturing NRCMs under hypoxia(1%O2,5%CO2,94%N2)for 10 hours.The senescence cell model was confirmed by SA-?-gal-staining.We also use the immunofluorescence to assess the number of p16INK4a-positive cardiomyocytes after hypoxia.Western blot was to detect the change of GATA4 and CCN1 in the hypoxia-treated cardiomyocytes as well as in the medium.4.It was reported that stress-induced senescent cells could secret CCN1,which was able to suppress the myofibroblast viability to possess antifibrosis effects in the liver.However,we still do not know whether a similar effect exists in the senescent cardiomyocytes.We isolated and cultured cardiac myofibroblasts as previously published protocol.We assessed the time-dependent effects of exogenous CCN1(5 ?g/mL)on myofibroblast viability by the CCK8 assay.Similarly,dose-dependent effects of CCN1 on myofibroblast vitality was also detected by CCK8 assay.Next,we re-introduced recombinant CCN1 protein to Gata4-shRNA mice by tail vein injection.We determined the heart functions after CCN1 administration in Gata4-shRNA mice using echocardiography.5.We previously verified that ALDH2 was closely related to cardiovascular disorders.Of note,almost 30-40%of the Asian population carries an inactivating point mutation in the ALDH2 gene,and this mutation is closely linked with myocardial infarction,stroke,as well as diabetes mellitus.But its correlation with cardiomyocyte senescence remains unknown.Herein,we cultured the H9C2 cell line under hypoxia for a different time and detected the expression and activity of ALDH2.To further answer the contribution of ALDH2,we treated the cells with ALDH2 activator,Alda-1,and detected the expression of senescence markers by RT-qPCR6.In vivo,we established the AMI models in both ALDH2 knockout and wild-type mice.After that,we used Western blot and immunofluorescence to compare the expression of p16INK4a,GATA4,and CCN1 between KO and WT mice after AMI.Furthermore,we used AAV9 to knockdown the GATA4 in ALDH2 knockout mice and compared the different responses between KO and WT mice.Results1.SA-?-gal staining identified that SA-?-gal-positive senescent cells accumulated both in the infarct and border areas 4 weeks after AMI surgeries.Masson staining showed that the SA-?-gal-positive senescent cells co-localized with the cardiomyocytes in the border areas.Histological analysis confirmed that senescence marker p16INK4a increased as early as 1 day and lasted for 4 weeks after LAD ligation.Additionally,another senescence marker p21CIP1/WAF1 was upregulated,beginning at 1-week postinfarction and lasting for 4 weeks after surgery.In summary,multiple senescence biomarkers accumulated in ischemic hearts indicating that cardiomyocyte senescence participated in the post-AMI hearts.2.In this study,we observed increased cardiomyocytes with damaged DNA based on y-H2AX immunostaining in postinfarction hearts.As the downstream of damaged DNA,GATA4 was raised accordingly in post-AMI myocardium in the border areas.To examine the effects of GATA4 inhibition on activated SASP,we utilized AAV9-Gata4-shRNA to achieve 50%GATA4 knockdown in vivo.Western blot revealed that the SASP was effectively activated after AMI because selected SASP factors including IL-la,TNF-a,MCP-1,and CCN1 were significantly increased after AMI.Compared with negative-control vectors(NC),AAV9-Gata4-shRNA significantly downregulated activated SASP factors IL-la,TNF-a,and CCN1.Taken together,these findings indicate that GATA4 accumulates in the damaged DNA after AMI and is required for postinfarction SASP activation.3.Echocardiography was utilized to assess heart function after mice were transfected with AAV9-Gata4-shRNA or negative control vectors.As data demonstrated,the impaired LV ejection fraction and fractional shortening were further deteriorated by AAV9-Gata4-shRNA 4 weeks after left anterior descending coronary artery ligation.Furthermore,the LV posterior wall at end-systolic became thinner after AAV9-Gata4-shRNA administration.Furthermore,the post-AMI hearts displayed LV dilatation as left ventricular end systolic,end-diastolic internal diameter and left ventricular end-diastolic volume were significantly increased.The dilated phenotype was further exaggerated by GATA4 knockdown.In addition,the collagen content was upregulated from 60%to 80%of the total lesion areas by Masson staining.These data suggested that GATA4 was required for post-AMI heart recovery,and GATA4-mediated SASP exerted protective effects in post-AMI functional restoration.4.We isolated and cultured RNCMs to recapitulate postinfarction cardiomyocyte senescence in vitro.The immunofluorescence staining verified>80%of cells as a-actin-positive cardiomyocytes.The senescence cell model was successfully established and was characterized by increased SA-?-gal-positive cells.Similarly,the number of p16INK4a-positive cardiomyocytes increased after hypoxia.Western blot confirmed a time-dependent GATA4 protein increase in vitro.Consistent with the in vivo observation that GATA4 was required for SASP activation,Gata4-small interfering RNAs significantly suppressed CCN1 increases in both cell extracts and supernatants.These data suggest that GATA4 is essential to the CCN1 expression in the senescent cardiomyocytes.5.The CCK8 assay demonstrated that exogenous CCN1 protein negatively regulated fibroblast viability in a time-dependent and dose-dependent pattern,corroborating previous reports that CCN1 directly downregulates fibroblast viability.Next,we re-introduced recombinant CCN1 protein to Gata4-shRNA-injected mice.Compared with the BSA control group,CCN1 administration effectively reserved cardiac function 4 weeks after surgery.Taken together,these results suggest that GATA4-related CCN1 secretion participates in the heart function preservation of senescent cardiomyocytes.6.We treated H9C2 cell line with hypoxia and observed an increase of senescence biomarkers p53 and a decreased ALDH2 activity.The pharmacological activation of ALDH2 by Alda-1 attenuated hypoxia-induced myocardial senescence.In vivo,compared with wild-type mice,ALDH2 knockout mice manifested increased p 16INK4a expression with unaltered GATA4 and CCN1 expression levels after AMI,via Western blot.In addition,immunofluorescence staining indicated a reduction of CCN1 in p16INK4a-positive senescent after AMI,compared with wild-type mice.Functionally,GATA4 inhibition was observed to alleviate the impaired heart function as ejection fraction was statistically restored after AMI in ALDH2 knock-out mice.Conclusions1.Myocardial senescence is involved in the ischemic heart tissues.2.Cardiomyocyte senescence was essential to postinfarction heart function via the GATA4-CCN1 pathway to exert antifibrosis effects after AMI.3.ALDH2 deficiency blocked GATA4-CCN1 pathway to influence the effects of cardiomyocyte senescence after AMI.SignificanceUnderstanding the precise mechanism behind cardiac senescence helps in the development of new drugs and in determining appropriate clinical strategies in the future.Understanding the diverse roles and mechanisms of cardiomyocyte senescence by ALDH2 is promising for improving cardiac remodeling in ALDH2 mutant-type people or in conditions of ALDH2 inactivation,such as nitrate tolerance and hyperglycemia.Regulating the GATA-binding factor 4-related pathway is promising for improving cardiac remodeling in aldehyde dehydrogenase2 mutant-type persons.