The Role Of Cathepsin B In Diabetic Cardiomyopathy

Background:In recent years,the incidence of cardiovascular disease in China has been rising,and it has become the biggest killer of people's health.It is worth noting that the sharp increase in the number of patients with diabetes is a very important reason for the high incidence of cardiovascular disease.According to the latest data from the International Diabetes Federation(IDF),approximately 463 million adults worldwide suffer from diabetes in 2019.It is expected that there will be 578 million diabetic patients by 2030,and will continue to grow to 700 million by 2045.In China,about 116 million adults suffer from diabetes in 2019,which still ranks first in the world.Diabetes is an independent risk factor for heart failure.Even if the two risk factors of coronary heart disease and hypertension are controlled,diabetes still increase the risk of heart failure.Therefore,Clinicians have attached more importance to diabetic cardiomyopathy(DCM).DCM is recognized as an independent primary disease that does not depend on hypertension,coronary artery disease and other known heart disease.DCM manifests as diastolic dysfunction in the early stage with decreased myocardial compliance and impaired diastolic filling,and systolic dysfunction in the late stage,which eventually develops into congestive heart failure.Although the current therapy to lower blood glucose or lipids and inhibit the excessive activation of neurohumoral system can improve the cardiac dysfunction caused by DCM to a certain extent,there are still a considerable number of patients with poor treatment effect,and the prevalence and mortality of DCM induced heart failure still remains high.Therefore,further elucidating the specific pathogenesis of DCM and finding effective prevention targets are of great significance for reducing the cardiovascular complications of diabetic and the development of heart failure.Cathepsin B(CTSB)is a cysteine endopeptidase that is widely expressed on lysosomes and belongs to the papain family.The CTSB gene is located on chromosome 8p22(22b)and contains 13 gene exons.It plays an important role in regulating innate immunity,extracellular matrix balance,inflammation and apoptosis.At present,most of studies about CTSB focus on the field of cancer.Studies have found that CTSB can promote the degradation of basement membrane,which is closely related to the invasion and metastasis of cancer cell.On the heart aspect,studies have reported that CTSB protein and mRNA levels are significantly increased in the heart of patients with dilated cardiomyopathy,and their expression levels are negatively correlated with Left ventricular ejection fraction(LVEF),but the specific mechanism is still unknown.CTSB is also closely related to the pathogenesis of atherosclerosis,myocarditis,myocardial infarction,and pressure overload induced cardiac remodeling.It can be seen that CTSB plays an important role in cardiovascular disease.However,the role of CTSB in DCM has not been reported.This study intends to use CTSB gene knockout and adeno-associated virus 9(AAV9)CTSB overexpressing mice and to investigate the role and pathogenic mechanism of CTSB in DCM,with a view to proposing new targets and ideas for the prevention and treatment of DCM.Methods:Part one:Animal experiment:C57BL/6 male mice aged 8 weeks and weighing 22.0g-25.9g were randomly divided into two groups:the DCM group and the control(Con)group.The DCM group was intraperitoneally injected with Streptozocin(STZ),50mg/kg/day,and the Con group was intraperitoneally injected with an equal volume of citric acid buffer for 5 days.After continued feeding for 16 weeks,heart tissues of the mice were harvested.Western blot was used to detect the protein expression of CTSB in myocardial tissue.Immunohistochemical staining was used to detect the localization and expression of CTSB in cardiomyocytes.Cell experiments:Primary rat cardiac myocytes(NRVM)were used in vitro experiments.The cells were divided into three groups:low glucose control group(Negative control,NC),low glucose hypertonic control group(Hyperosmosis,HO)and High glucose(HG),and treated with media containing different concentrations of glucose and/or mannitol.The processed NRVM were collected 24 hours later,the protein was extracted and the protein expression of CTSB in each group was detected by Western Blot.Immunofluorescence staining was used to detect the localization and expression of CTSB in NRVM.Part two:Animal experiments:1.C57BL/6 wild type and CTSB gene knockout male mice aged 8 weeks,weighing 22.0g-25.9g were divided into 4 groups:wild type control group(WT-Con),CTSB gene knockout control group(KO-Con),wild type DCM group(WT-DCM),CTSB gene knockout DCM group(KO-DCM).2.C57BL/6 wild-type male mice aged 8 weeks and weighing 22.0g-25.9g were used,adeno-associated virus 9 CTSB tail vein injection was performed to overexpress CTSB.The virus injection time was 10 weeks after the injection of STZ.The experiment was divided into 4 groups:AAV9 NC control group(AAV9 NC-Con),AAV9 CTSB overexpression control group(AAV9 CTSB-Con),AAV9 NC DCM group(AAV9 NC-DCM),AAV9 CTSB overexpression DCM group(AAV9 CTSB-DCM).The DCM group was intraperitoneally injected with STZ,50mg/kg/day,and the Con group was intraperitoneally injected with an equal volume of citric acid buffer for 5 consecutive days.The blood glucose and body weight of mice were measured at 1 week,4 weeks,8 weeks,12 weeks and 16 weeks after STZ injection.Echocardiography and haemodynamic measurements was performed to detect the heart function of mice in each group at 16 weeks,and the hearts of the mice were harvested.Cardiac fibrosis and inflammation were detected by Picric sirius red(PSR)staining,CD45,and CD68 immunohistochemical staining.Real-time quantitative PCR was used to detect the mRNA expression levels of fibrosis and pro-inflammatory related markers.TUNEL and Caspase-1 immunohistochemical staining,Caspase-1 activity,Gastermin D,Gastermin D P30 protein expression,Caspase-1,IL-1?,IL-18 mRNA expression levels were detected to observe the pyroptosis in the mice heart of each group.Cell experiments:1.CTSB small interfering RNA was used to transfect NRVM to silence CTSB.The cells were divided into four groups:Blank control group(si NC+HO),CTSB silence control group(si CTSB+HO),Blank high glucose Group(si NC+HG),CTSB silence high glucose group(si CTSB+HG).2.Adenovirus(Adenovirus,Ad)was used to transfect NRVM to overexpress CTSB.The cells were divided into four groups:Blank control group(Ad NC+HO),CTSB overexpression control group(Ad CTSB+HO),Blank high Glucose group(Ad NC+HG),CTSB overexpression high glucose group(Ad CTSB+HG).The control group was stimulated by 5.5 mM glucose+27.5 mM mannitol for 24 hours,and the high glucose group was stimulated by 33 mM glucose for 24 hours.Hoechst 33342/PI staining,TUNEL staining,Caspase1 immunofluorescence staining,the protein expression levels of Gastermin D and Gastermin D P30,Caspase-1 activity,LDH release,the mRNA expression levels of Caspase-1,IL-1?,and IL-18 were detected to observe the pyroptosis of NRVM in each group.Part three:Animal experiments:1.C57BL/6 wild type and CTSB gene knockout male mice aged 8 weeks,weighing 22.0g-25.9g were divided into four groups:WT-Con group,KO-Con group,WT-DCM group,KO-DCM group.2.C57BL/6 wild-type male mice aged 8 weeks and weighing 22.0g-25.9g were used and adeno-associated virus 9 CTSB tail vein injection was performed to overexpress CTSB.The virus injection time was 10 weeks after the injection of STZ.The experiment was divided into 4 groups:AAV NC-Con group,AAV9 CTSB-Con group,AAV NC-DCM group,AAV9 CTSB-DCM group.The diabetic cardiomyopathy group was intraperitoneally injected with STZ,50 mg/kg/day,and the control group was intraperitoneally injected with an equal volume of citric acid buffer for 5 days.16 weeks later,the hearts were harvested and myocardial tissue proteins were extracted.Western Blot was used to detect the expression levels of NLRP3,ASC,and Pro-caspase-1 in each group.Cell experiments:1.CTSB small interfering RNA was used to transfect NRVM to silence CTSB.The cells were divided into four groups:si NC+HO group,si CTSB+HO group,si NC+HG group,si CTSB+HG group.2.Adenovirus was used to transfect NRVM to overexpress CTSB.The cells were divided into four groups:Ad NC+HO group,Ad CTSB+HO group,Ad NC+HG group,and Ad CTSB+HG group.The control group was treated with 5.5mM glucose+27.5mM mannitol for 24 hours,and the high glucose group was treated with 33mM glucose for 24 hours.Western Blot was used to detect the protein expression levels of NLRP3,ASC and Pro-caspase-1 in each group.3.The NRCMs were divided into two groups:the control group(HO)and the high glucose group(HG).The control group was treated with 5.5mM glucose+27.5mM mannitol for 24 hours,and the high glucose group was treated with 33mM glucose for 24 hours,the interaction between CTSB and NLRP3 in NRVM was detected by immunoprecipitation.Results:Part one:1.CTSB expression was significantly increased in the heart of DCM mice induced by STZ.2.CTSB was mainly distributed in the cytoplasm around the nucleus of NRVM,and the expression of CTSB in NRVM was significantly increased after high glucose stimulation.Part two:1.One week after the injection of STZ,the blood glucose of the mice in the DCM group increased significantly,and the body weight of the mice in the DCM group began to gradually decrease at 12 weeks.However,there was no significant difference in blood glucose and body weight between the WT-DCM group KO-DCM group at each time node.There was also no significant difference in blood glucose and body weight between the AAV9 NC-DCM group AAV9 CTSB-DCM group at each time node.2.Echocardiography and hemodynamics results showed that the left ventricular end-systolic diameter,left ventricular end-diastolic diameter was increased,LVEF,fractional shortening,dp/dt max and dp/dt min were reduced significantly in DCM mice.CTSB gene knockout can significantly improve the cardiac dysfunction,and CTSB overexpression can further worsen the cardiac dysfunction in DCM mice.3.The degree of fibrosis,CD45 and CD68 labeled inflammatory cell infiltration,mRNA expression levels of fibrosis and inflammation-related markers,such as Collagen ?,Collagen ?,CTGF,TNFa,MCP-1 and IL-6were significantly increased in DCM mice,while the degree of fibrosis and inflammation were alleviated after CTSB gene knockout and were aggravated significantly after CTSB overexpression in DCM mice.4.Proportion of TUNEL positive cells,protein expression levels of Gastermin D and Gastermin D P30,Caspase-1 enzyme activity,and mRNA expression of Caspase1,IL-1?,IL-18 increased significantly in DCM mouse heart,while these indicators decreased significantly after CTSB gene knockout,and increased further after CTSB overexpression in the DCM mouse heart.5.After high glucose stimulation,the number of PI and TUNEL positive cells,the proteins expression levels of Gastermin D and Gastermin D P30,Caspase-1 enzyme activity,LDH release in cell supernatants,the mRNA expression levels of Caspase-1,IL-1? and IL-18 were significantly increased,and these indexes were significantly decreased after CTSB silencing,but increased further after CTSB overexpression.Part three:1.Both in vivo and in vitro experiments showed that CTSB deletion inhibited the activation of NLRP3 inflammasome with the decreased protein expression levels of NLRP3,ASC and Pro-caspase-1 under the stimulation of high glucose.2.Both in vivo and in vitro experiments showed that CTSB overexpression can increase the activation of NLRP3 inflammasome with the further increased protein expression levels of NLRP3,ASC and Pro-caspase-1 under high glucose stimulation.3.In the basal state,there was a small amount binding between CTSB and NLRP3 in NRVM.Under high glucose stimulation,the binding between CTSB and NLRP3 increased.Conclusion:1.The expression of CTSB was significantly increased in DCM and high glucose stimulated NRVM.2.CTSB gene knockout could alleviate high glucose induced myocardial fibrosis,inflammation and pyroptosis.3.CTSB overexpression could aggravate high glucose-induced myocardial fibrosis,inflammation and pyroptosis.4.CTSB might activate NLRP3 inflammasome by combining with NLRP3.