Effects And Mechanisms Of Hydrogen Sulfide In Diabetic Cardiomyopathy

Objective Diabetic cardiomyopathy（DCM）,a common diabetic complication,is the main cause of increased mortality in diabetic patients,which lacks effective clinical methods.Hydrogen sulfide（H₂S）as one kind of gasotransmitter is mainly produced under the catalysis of cystathionine-γ-lyase（CSE）in cardiovascular system.However,the effect of H₂S on diabetic cardiomyopathy is unclear.Our study was to investigate the real role and underlying mechanisms of H₂S in diabetic cardiomyopathy,and to provide novel ideas for the prevention and treatment of diabetic cardiomyopathy.Methods Male 8-week-old C57BL/6（wild type,WT）mice and CSE knockout（KO）mice were injected intraperitoneally with streptozocin（STZ,60 mg/kg/d）in sodium citrate buffer for 5 consecutive days,and the same dosage of sodium citrate buffe was administrated in control group.Male 8-week-old C57BL/Ks J-db/dm（WT）and C57BL/Ks J-db/db（db/db）mice were intraperitoneally injected with H₂S donor sodium hydrosulfide hydrate（Na HS,50μmol/kg/d）for 12consecutive weeks,and the same dosage of normal saline was administrated in control group.During the experiment,fasting blood glucose（FBG）was monitored regularly.After 12 weeks,H₂S level in plasma,H₂S production and CSE m RNA level in myocardium were detected.Cardiac function was evaluated by echocardiography.The degree of myocardial hypertrophy and fibrosis was assessed by hematoxylin-eosin（HE）,wheat germ agglutinin（WGA）,Sirus-red and Masson staining.The content of lactate dehydrogenase（LDH）in serum and adenosine triphosphate（ATP）in myocardium were measured.Levels of oxidative stress in myocardium were assessed by dihydroethidium（DHE）,2,7-dichlorodihydrofuorescein diacetate（DCFH-DA）and Mito SOX staining.The mitochondrial ultrastructure of myocardium was observed by transmission electron microscopy.Apoptosis was detected by Td T-mediated d UTP nick end labeling（TUNEL）staining.The expression of cysteinyl aspartate specific proteinase 3（Caspase 3）,receptor interacting protein kinase 1（RIPK1）,receptor interacting protein kinase 3（RIPK3）,mixed lineage kinase domain like protein（MLKL）,NOD-like receptor pyrin domain containg 3（NLRP3）,cysteinyl aspartate specific proteinase 1（Caspase 1）and interleukin-1β（IL-1β）in myocardium was detected by western blot.Primary cardiomyocytes were cultured from newborn Sprague-Dawley（SD）rats and were pretreated with CSE inhibitor DL-Propargylglycine（PAG,3 mmol/L）or Na HS（50μmol/L）for 4 h with normal glucose（NG,5.5 mmol）or high glucose（HG,33.3mmol/L）for 48 h.In cardiomyocytes,LDH release and ATP levels were detected.Oxidative stress were evaluated by DHE staining and Mito SOX staining.Mitochondrial membrane potential was assessed by JC-1staining.Apoptosis were detected by TUNEL staining.Caspase 3,RIPK1,RIPK3,MLKL,NLRP3 and Caspase 1 expression were measured by immunofluorescence and western blot.Results 1.The plasma H₂S level,myocardial H₂S production and CSE m RNA expression were decreased in diabetic mice,suggesting that H₂S production was impaired in diabetic mice.2.Compared with WT diabetic mice,the cardiac function was further exacerbated.Myocardial hypertrophy,myocardial fibrosis and myocardial injury were aggravated.Myocardial oxidative stress and mitochondrial damage were promoted.The number of TUNEL staining positive cells was increased.The expression of necroptosis-related proteins including Cleaved caspase 3,RIPK1,RIPK3 and p-MLKL as well as inflammation-related proteins including NLRP3,Caspase 1 p20 and IL-1βwere further up-regulated in CSE KO mice after STZ injection.3.CSE inhibitor PAG further aggravated cell damage and oxidative stress,reduced mitochondrial membrane potential,increased the number of TUNEL positive cells,and up-regulated Cleaved caspase 3,RIPK1,RIPK3,p-MLKL,NLRP3 and Caspase 1 protein expression in cardiomyocytes with high glucose stimulation.4.Na HS had no significant influence on blood glucose in db/db mice.However,Na HS improved cardiac function,attenuated myocardial hypertrophy,myocardial fibrosis and myocardial injury,suppressed myocardial oxidative stress and mitochondrial damage,reduced the number of TUNEL staining positive cells and decreased protein expression of Cleaved caspase 3,RIPK1,RIPK3,p-MLKL,NLRP3,Caspase 1 p20 and IL-1βin db/db mice.5.Na HS alleviated cell damage and oxidative stress,increased mitochondrial membrane potential,decreased the number of TUNEL positive cells,and reduced the expression of Cleaved caspase 3,RIPK1,RIPK3,p-MLKL,NLRP3 and Caspase 1 in cardiomyocytes with high glucose stimulation.Conclusion H₂S production was impaired in diabetic mice.H₂S deficiency aggravated mitochondrial damage,increased ROS accumulation,promoted necroptosis,activated the NLRP3 inflammasome,and finally exacerbated diabetic cardiomyopathy.Exogenous H₂S supplementation alleviated necroptosis to suppress NLRP3inflammasome activation and attenuate diabetic cardiomyopathy via mitochondrial dysfunction improvement and oxidative stress inhibition.