Cardiomyocyte-specific Knockdown Of Sirt6 In The Pathogenesis Of Diabetic Cardiomyopathy

BackgroundDiabetic cardiomyopathy(DCM)defined by diastolic and systolic dysfunction independently of overt clinical coronary artery disease,hypertension,valvular disease and other conventional cardiovascular risk,eventually develops into heart failure,which is strongly associated with a high incidence of mortality in people with diabetes mellitus(DM).Because of its complex and unclear pathogenesis,there is a lack of uniform and effective specific therapeutic measures.Therefore,it is urgent to explore the pathogenesis associated with diabetic cardiomyopathy.Although studies have now suggested that silent information regulator 6(Sirt6)is a therapeutic target for improving DCM,the specific mechanism remained to be clarified.Apoptosis and Nucleotide binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome mediated pyroptosis have been demonstrated to play an important role in the occurrence and development of DCM.In addition,studies have revealed that activation of AMP-activated protein kinase(AMPK)can inhibit apoptosis and NLRP3 inflammasome mediated pyroptosis by increasing autophagy.However,it is not clear whether Sirt6 is involved in regulation of apoptosis and NLRP3 inflammasome via the AMPK-dependent effect in DCM.Accordingly,we investigated the mechanism and effects of Sirt6 on apoptosis and the NLRP3 inflammasome in DCM.Our research is aimed to provide a novel therapeutic strategy for the treatment of DCM.Objective1.To elucidate that cardiac-specific knockdown of Sirt6 exacerbates high glucose and high fat-induced cardiac dysfunction in mice.2.To elucidate that cardiac-specific knockdown of Sirt6 exacerbates high glucose and high fat-induced cardiac dysfunction by exacerbating cardiomyocyte apoptosis and pyroptosis.Methods1.Construction of cardiomyocyte-specific Sirt6 knockout mice:cardiomyocyte-specific Sirt6 knockout mice(Sirt6 CKO)were generated by crossing a mouse line carrying conditional allele of Sirt6(Sirt6 fl/fl)and Myh6-Cre mice.2.Construction of DCM model:Eight-week-old Sirt6 CKO mice and their wild-type(WT)mice with C57BL/6 background were randomly divided into diabetes mellitus(DM)group and control group.the DM group were fed with high-fat diet(HFD;60 kcal%fat,TP23520;Trophic Diet)for three months to induce insulin resistance,followed by a single intraperitoneal injection of streptozotocin(STZ;pH 4.5)at 100 mg/kg in 0.1 mol/L of freshly prepared sodium citrate buffer to induce partial insulin deficiency.Seven days after STZ injection,mice with fast blood glucose(FBG)higher than 250 mg/dL were considered as diabetic.Age-matched mice were fed with normal diet for 3 months,followed by an injection of vehicle solution(0.1 mol/L of citrate acid buffer,pH 4.5)instead of STZ,which were defined as Control.Mice in the DM and control groups continued to be fed high-fat and normal diets for 12 weeks before further experiments.3.Echocardiography was performed to measure left ventricular ejection fraction(LVEF)and left ventricular shortening fraction(FS)to evaluate cardiac systolic function.4.All mice were euthanized and then HE staining was performed to observe the structural changes of myocardial tissue.Masson staining kit and Sirius Red staining kit were applied to assess the collagen deposition and fibrosis level of myocardial interstitium.The expression of cleaved caspase3 in myocardial tissues was observed by immunochemical tissue staining.5.Extraction of primary neonatal rat cardiomyocytes:Sprague-Dawley rats(1-3 days old)were washed and sterilized by alcohol immersion,and then the hearts of neonatal rats were quickly removed,rinsed,cut into pieces and digested with collagenase Ⅱ.The supernatant was collected,centrifuged,filtered,and the digestion was terminated.According to the different attach speed,cardiomyocytes and fibroblasts were separated.The isolated cardiomyocytes were inoculated onto well plates for subsequent experiments.6.Primary cardiomyocyte intervention:The expression of Sirt6 in cardiomyocytes under high glucose stimulation was detected by western Blot.Subsequently,NRCMs were transfected with Sirt6 overexpressing adenovirus to overexpress Sirt6.7.The apoptosis rate of cardiomyocytes was detected by applying the apoptosis flow cytometry kit.8.The protein expression levels of cleaved caspase3,NLRP3,cleaved caspase1,cleaved IL-1β and GSDMD-N in DCM myocardial tissue and in high glucose stimulated cardiomyocytes were detected by western blot.9.The expression levels of autophagy-related proteins P62,LC3B II,and AMPK phosphorylation in high glucose stimulated cardiomyocytes were detected by western blot.10.We applied AMPK inhibitor Compound C to NRCMs,and then the expression levels of P62,LC3B Ⅱ,T-AMPK,P-AMPK,cleaved caspase3,NLRP3,cleaved caspase1,and cleaved IL-1β protein were detected by western blot.Results1.Compared with Control group,the expression of Sirt6 in the myocardial tissue of DM group was significantly reduced,accompanied by promoted disorder of the myocardial structure and cardiac fibrosis,which aggravated the cardiac functional impairment in DCM.The aforementioned deteriorations were further exacerbated in the diabetic SIRT6 CKO mice,indicating that cardiac-specific knockout of Sirt6 promoted the progress of DCM.2.Compared with Control group,the expressions of apoptosis-related protein cleaved caspase3 and pyroptosis related protein such as NLRP3,cleaved caspase-1,cleaved IL-1β and GSDMD-N in the myocardial tissue of DM group were significantly increased.After specific knockdown of Sirt6 in cardiomyocytes using the Cre-loxp system,the expression levels of these apoptosis and pyroptosis related proteins were further significantly increased.3.Under high glucose stimulation,the expression of Sirt6 in NRCMs is downregulated,accompanied by increased apoptosis and pyroptosis of cardiomyocytes,as well as inhibition of AMPK pathway and autophagy.Sir6 overexpression significantly inhibited cardiomyocyte apoptosis and pyroptosis,as well as increased AMPK phosphorylation and autophagy.while when we applied AMPK inhibitor Compound C to NRCMs,Compound C not only significantly reduced AMPK phosphorylation and autophagy,but also reversed decrease of apoptosis and pyroptosis in NRCMs by overexpressing Sirt6.ConclusionThe results showed that cardiomyocyte-specific knockdown of Sirt6 exacerbated cardiac remodeling and myocardial apoptosis and pyroptosis in DCM,thereby exacerbating cardiac dysfunction.In vitro experiments,our results showed that Sir6 overexpression significantly inhibited high glucose-induced cardiomyocyte apoptosis and pyroptosis,as well as significantly increased AMPK phosphorylation and autophagy,whereas administration of the AMPK inhibitor Compound C reversed decrease of apoptosis and pyroptosis in NRCMs by overexpress Sirt6.In conclusion,Sirt6 inhibits cardiomyocyte apoptosis and NLRP3 inflammatory inflammasome-mediated pyroptosis by stimulating AMPK phosphorylation and increasing autophagy,thus providing a novel strategy and target for treating DCM.