Protective Effects Of Resveratrol On Doxorubicin-induced Cardiotoxicity By SIRT1-SESN2 Pathway

BackgroundDoxorubicin(DOX)is one of the most widely used and effective anthracycline anticancer drugs.However,the cardiotoxicity caused by DOX seriously affects the quality of life of cancer survivors and limits its wide clinical application.To date,there is no effective targeted therapeutic strategy,neither prophylactic nor curative,to protect against DOX-induced cardiotoxicity.Resveratrol(RES)is a natural polyphenolic compound and exerts antioxidant,anti-inflammatory and anticancer properties.Preclinical and clinical studies have shown that RES can not only prevent cardiac diseases,but also enhance the anticancer effect of DOX.Therefore,RES has a broad application prospect in the prevention and treatment of DOX-induced cardiotoxicity.However,the mechanisms underlying the beneficial effects of RES are not totally elucidated.Although RES is a well-known potent activator of sirtuin 1(SIRT1),strong evidence has shown that RES could also regulate a variety of other signaling pathways to perform its biological functions,suggesting that RES exerts its protective role through both SIRT1-dependent and SIRT1-independent signaling pathways.Therefore,in the present study,the specific molecular mechanisms underlying the beneficial effects of RES in DOX-induced cardiotoxicity,particularly its dependence of SIRT1,remain to be further clarified.AMP-activated protein kinase(AMPK)is a key downstream target of the SIRT1 signaling,which exerts a beneficial role in the treatment of cardiac diseases.Furthermore,sestrin 2(SESN2),a conserved stress-induced protein,has been proved to be a positive regulator of AMPK activation in the heart and decreased in DOX-induced chronic cardiomyopathy and dilated cardiomyopathy.Overexpression of SESN2 ameliorated DOX-induced mitochondrial dysfunction and mitochondrial autophagy disorder.In contrast,genetic ablation of Sesn1 and Sesn2 could aggravate DOX-induced cardiac pathologies,which was associated with the suppression of the AMPK signaling.However,whether SESN2 mediates the cardioprotective effects of SIRT1 on AMPK activation in DOX-treated cardiomyocytes has not been identified.Objectives1.To verify whether the protective effects of RES in DOX-induced cardiotoxicity depends on SIRT1 signaling.2.To explore whether SESN2 mediates the cardioprotective effects of SIRT1 on AMPK activation in DOX-treated cardiomyocytes.3.To investigate the mechanism by which SIRT1 regulates SESN2 activation in cardiomyocytes.MethodsSirt1flox/flox mice and cardiac-specific Sirt1 knockout Sirt1flox/flox Myh6-creEsr1,Sirt1-CKO)mice were randomly divided into eight groups.DOX or the same volume of vehicle(saline)was intraperitoneally injected once a week with a dose of 5 mg/kg for 4 weeks.RES(10 mg/kg/day)or the same volume of vehicle(saline)was intraperitoneally injected every day for 5 weeks.Cardiac function was examined by echocardiography.FITC-conjugated wheat germ agglutinin(FITC-WGA)staining was used to detect myocyte cross-sectional areas.Cardiac collagen deposition was examined by Masson’s trichrome staining and Sirius red staining.Relative mRNA levels of atrial natriuretic peptide(Anp),brain natriuretic peptide(Bnp),connective tissue growth factor(Ctgf),tumor necrosis factor-α(Tnfa),interleukin-1β(Il1b),catalase(Cat)and superoxide dismutase(Sod)in mice hearts and H9c2 cells were detected by real-time quantitative polymerase chain reaction(RT-qPCR).Cardiac expression of TNF-α,4-hydroxynonenal(4-HNE)and 3-nitrotyrosine(3-NT)was examined by immunohistochemical(IHC)staining.Nuclear expression of nuclear factor-κB(NF-κB)p65 and nuclear factor erythroid 2-related factor 2(NRF2)as well as the expression of heme oxygenase-1(HO-1)and NAD(P)H quinone dehydrogenase 1(NQO1)in mice hearts were detected by immunofluorescent(IF)staining.Reactive oxygen species(ROS)levels in mice hearts and H9c2 cells were assessed by dihydroethidium(DHE)staining,2’,7’-dichlorofluorescein diacetate(DCFH-DA)staining and flow cytometry.Apoptotic rate of cardiomyocytes was detected using Terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)staining and flow cytometry.The protein expression of nuclear NF-κB p65,nuclear NRF2,HO-1,SIRT1,SESN2,P-AMPKα,AMPKα,phosphorylated mammalian target of rapamycin(P-mTOR),P-S6 ribosomal protein(P-S6),S6,cleaved caspase-3 and murine double minute 2(MDM2)in mice hearts,H9c2 cells and primary cardiomyocytes was detected by western blot.The ubiquitin levels of SESN2 and protein-protein interaction between SESN2 and MDM2 in hearts,primary cardiomyocytes and H9c2 cells were determined by immunoprecipitation.Results1.The protective effects of RES on DOX-induced cardiotoxicity were dependent on SIRT11.1 Identification of cardiac-specific Sirt1 knockout miceThere was almost no expression of SIRT1 protein in cardiac tissues of Sirt1-CKO mice.1.2 RES ameliorated DOX-induced cardiac dysfunction via SIRT1To explore the role of SIRT1 in DOX-induced cardiotoxicity,echocardiography was performed to examine cardiac function.The results demonstrated that compared with Ctrl group,DOX application induced cardiac dysfunction as evidenced by decreased ejection fraction and fractional shortening and increased systolic left ventricular internal dimension and systolic left ventricular volume in Sirt1flox/flox mice.These changes were more pronounced in Sirtl-CKO DOX group compared with Sirt1flox/flox DOX group,whereas RES treatment significantly improved DOX-induced cardiac dysfunction in Sirt1flox/flox mice,but not in Sirt1-CKO mice.1.3 RES inhibited DOX-induced myocardial remodeling via SIRT1Cardiomyocyte size was increased in response to DOX by analysis of myocyte area via FITC-WGA staining.Furthermore,Masson’s trichrome and Sirius red staining showed collagen accumulation in DOX-impaired mice hearts.Morphological hypertrophy and fibrosis of hearts were further confirmed by elevated mRNA levels of hypertrophic markers Anp and Bnp,and fibrotic marker Ctgf,respectively.All of these indices were more significantly increased in Sirt1-CKO DOX group than Sirt1flox/flox DOX group,and almost completely inhibited by RES treatment only in Sirt1flox/flox mice.1.4 RES alleviated DOX-induced cardiac inflammatory response via SIRT1TNF-α protein and Il1b mRNA levels were increased in DOX groups of Sirt1fiox/flox and Sirt1-CKO mice.RES treatment prevented DOX-associated inflammation only in Sirt1fiox/flox mice.Moreover,DOX significantly increased the nuclear expression of NF-κB p65,and this effect was abolished by treatment with RES only in Sirt1fiox/flox mice.These results were further confirmed by in vitro studies with H9c2 cells.1.5 RES inhibited DOX-induced cardiac oxidative stress via SIRT1The fluorescence intensity of DHE was evident in cardiac tissues of Sirt1fiox/flox DOX group and further elevated in Sirt1-CKO DOX group.Moreover,cardiac oxidative damage,assessed by examining the levels of 4-HNE and 3-NT,was noted in Sirt1fiox/flox DOX group and further increased in Sirt1-CKO DOX group.RES treatment prevented all these effects in Sirt1fiox/flox mice,but not in Sirt1-CKO mice.In addition,RES treatment blocked DOX-induced downregulation of nuclear NRF2 and preserved its transcriptional activity,as confirmed by increased protein levels of HO-1 and NQOl and mRNA levels of Cat and Sod.However,the beneficial effects of RES were abolished in the cardiac tissues of Sirt1-CKO mice.These results were further confirmed by in vitro studies with H9c2 cells.1.6 RES reduced DOX-induced cardiomyocyte apoptosis via SIRT1Increased numbers of TUNEL-positive cells were observed in hearts of Sirt1fiox/flox DOX group and further increased in Sirt1-CKO DOX group as detected by TUNEL staining.RES treatment prevented this effect only in Sirt1fiox/flox DOX group.These results were further confirmed by in vitro studies with H9c2 cells.2.The activation of SESN2 and AMPKα by RES to improve DOX-induced cardiotoxicity was in a SIRT1-dependent mannerWestern blot analysis showed that the expression of SESN2 and P-AMPKa was significantly inhibited following DOX stimulation.Moreover,the knockout of Sirtl hindered RES-upregulated SESN2 and P-AMPK expression in DOX-impaired mice hearts.Moreover,P-mTOR and P-S6 levels were decreased in DOX-treated mice,and RES treatment did not apparently change DOX-suppressed mTOR activity both in Sirt1fiox/flox and Sirtl-CKO mice.The results of H9c2 cell experiments were consistent with the data in vivo.Subsequently,we confirmed the regulatory effects of SIRT1 on SESN2 and AMPKa by transfection with pcDNA3.1-Sirt1 plasmid.SIRT1 upregulation increased SESN2 and P-AMPKa expression in primary cardiomyocytes treated with DOX.3.SESN2 is a scaffold protein for SIRT1 and AMPKα in response to DOX injurySesn2 knockdown not only abolished the ability of RES on AMPKa activation but also significantly blocked RES-exerted antioxidative and anti-apoptotic properties in DOX-treated cardiomyocytes,whereas overexpression of SESN2 could largely prevent DOX-impaired AMPKa activation and DOX-induced oxidative stress and apoptosis.However,downregulation or upregulation of SESN2 had no apparent change on the expression of SIRT1,suggesting that SIRT1 activated AMPKa to improve DOX-induced cardiotoxicity via upregulation of SESN2.4.SIRT1 reduced DOX-induced SESN2 ubiquitination possibly by disturbing the interaction of SESN2 and MDM2DOX stimulation increased SESN2 ubiquitination,which was restored by RES in H9c2 cells.Further studies indicated that overexpression of SIRT1 by RES and pcDNA3.1-Sirt1 plasmid could disturb DOX-enhanced interaction of SESN2 and MDM2 both in primary cardiomyocytes and H9c2 cells.However,SIRT1 deficiency abolished RES-reduced interaction of SESN2 and MDM2 both in DOX-treated mice hearts and H9c2 cells.Furthermore,knockdown of Mdm2 reversed DOX-induced SESN2 downregulation in primary cardiomyocytes.Taken together,all these results implied the activation of SIRT1 could reduce DOX-induced SESN2 ubiquitination possibly through disturbing the interaction of SESN2 and MDM2 in cardiomyocytes.Conclusions1.RES could activate SIRT1 to improve DOX-induced cardiac structural and functional abnormalities through preventing oxidative damage and apoptosis.2.The benefits of SIRT1 activation by RES in DOX-induced cardiotoxicity required the activation of SESN2-mediated antioxidant and anti-apoptotic signals.3.SIRT1 activation by RES reduced DOX-induced SESN2 ubiquitination to upregulate SESN2 expression possibly by disturbing the interaction of SESN2 and MDM2.4.SIRT1 activation by RES increased phosphorylation of AMPKα via upregulation of SESN2.5.The cardioprotective effect of SIRT1 activation by RES was independent of mTOR in response to DOX-induced toxicity.