Monocytes-derived S100A9 Promotes Mitochondrial Dynamics Abnormalities And Metabolic Reprogramming In Diabetic Cardiomyopathy

Objective:Diabetes mellitus is a leading public health concern globally and the major risk factor for cardiovascular diseases with its morbidity consistently increasing.Diabetic cardiomyopathy is one of the cardiovascular complications associated with diabetes,but the incidence of diabetes-related heart failure remains high with tight glycemic control,so it is urgent to explore the other potential pathogenesis.Systemic inflammatory activation is a core driver of histopathological injury for multiple cardiovascular diseases,but whether inflammatory activation is an alternative therapeutic target in diabetic cardiomyopathy remains unclear.Mitochondrial quality control is a key regulatory mechanism for the balance of energy metabolism,and mitochondrial homeostasis is one of the important interventions for heart failure.How the mitochondrial quality control mechanism responds to diabetes-induced metabolic damage in diabetic cardiomyopathy remains to be questioned.Therefore,this study focused on the influence of sustained inflammatory activation induced by diabetes on mitochondrial dynamics of myocardial tissue,to explore the specific regulatory mechanism and whether targeting inflammatory myocardial injury is a feasible therapeutic strategy for intervention in diabetic cardiomyopathy.Methods:Transcriptomic analysis of the GEO database was used to screen potential target genes.Two animal models of diabetic cardiomyopathy,streptozotocin combined with high-fat diet and db/db spontaneous diabetes,were established.Langendorff perfusion and enzymatic digestion were used to isolate primary myocardial cells from diabetic mice and neonatal rats,respectively.The expression and localization of S100A9 in cardiac tissue and primary cardiomyocytes were verified by western blot,double-immunofluorescence staining and colocalization analysis.Flow cytometry and ELISA were used to detect proinflammatory monocyte subsets and S100A9 secretion levels in the peripheral blood of diabetic cardiomyopathy mice.Co-culture of the Transwell system was used to investigate the interaction between THP-1 monocytes and AC16 cardiomyocytes.In AC16cardiomyocytes,the activation of STAT3 by S1009 was screened and verified by TF activation profiling plate array and electrophoretic mobility shift assay（EMSA）.Protein-protein docking,co-immunoprecipitation,immunoprecipitation combined mass spectrometry,and truncation mutant plasmids overexpression were used to explore the key sites of STIP1 binding to STAT3.MFF-dependent mitochondrial fission was detected by proteomics,dual-luciferase reporter assay,Mito-tracker staining,and STAT3 gene editing.Metabolite changes in cardiomyocytes were detected by metabolomics,and metabolic reprogramming of cardiomyocytes was explored by integrative metabolomics-proteomics analysis.Paquinimod,the S100A9 specific inhibitor,was administrated to animal models of diabetic cardiomyopathy.HE staining,WGA staining,Masson-trichrome staining,Sirius Red staining,TUNEL staining,transthoracic echocardiography,transmission electron microscopy,metabolic cage analysis,mitochondrial isolation,western blot,and ELISA were performed to detect myocardial remodeling,cardiac function,mitochondrial function and myocardial metabolism changes in S100A9-targeted diabetic cardiomyopathy mice.Results:S100A9 was identified as a novel upregulated gene in DCM by transcriptomics in the GEO database.In heart tissues and primary mouse cardiomyocytes of db/db mice,S100A9 expression increased and co-localized to myocardial cells.In diabetic cardiomyopathy mice（STZ induction or db/db）,the pro-inflammatory CD11b⁺Ly6C^highCCR2⁺monocytes subset was dramatically increased and alleviated by paquinimod administration（the specific S100A9 inhibitor）.Co-culture assay of S100A9-knocked THP-1 monocytes with AC16 cardiomyocytes revealed that the high expression of S100A9 in cardiomyocytes under hyperglycemia resulted from secreted S100A9 derived from monocytes.Transcription factor profiling array and EMSA analysis proved that S100A9 functioned by activating STAT3.Protein-protein docking,co-immunoprecipitation,IP-MS analysis,and overexpression of STIP1（Δ259-427）truncation mutants revealed STIP1 bound STAT3 by TPR 5-8 domain and the binding could be promoted by S100A9.Proteomics,dual-luciferase reporter assay,Mito-tracker staining and STAT3 gene editing indicated STAT3 activation promoted MFF and FIS1 transcription and aggravated excessive mitochondrial fission.Citric acid was identified the hub metabolite of S100A9/STAT3intervention in AC16 cardiomyocytes by metabolomics.The integrative metabolomics-proteomics analysis revealed the inhibition of the TCA cycle and mitochondrial OXPHOS by S100A9 promoted metabolic reprogramming in cardiomyocytes for adaptive regulation.The disturbance of mitochondrial quality control and mitochondrial dysfunction induced by S100A9 in diabetes further aggravated myocardial injury.Myocardial staining,transmission electron microscopy,metabolic cage analysis,western blotting,and ELISA results all indicated that targeting the S100A9/STAT3/MFF signaling pathway with paquinimod improved myocardial remodeling,cardiac dysfunction,excessive mitochondrial fission and myocardial metabolism abnormalities in diabetic cardiomyopathy mice.Conclusions:In diabetes mellitus,proinflammatory monocytes were activated to promote the secretion of S100A9.By promoting the interaction between STIP1 and STAT3,S100A9activated cardiomyocyte STAT3,promoted MFF transcription,and triggered excessive mitochondrial fission.At the same time,the citric acid level was upregulated,TCA cycle and mitochondrial OXPHOS were suppressed.Ultimately,abnormal mitochondrial quality control and mitochondrial dysfunction aggravated DCM,and targeting S100A9/STIP1/STAT3/MFF-dependent mitochondrial fission pathway by paquinimod might be an effective treatment strategy for DCM.