Role Of DNMT3A-Mediated MTHFR Methylation In Diabetic Cardiac Fibrosis

Diabetic myocardial fibrosis,a pathological process triggered by long-term pathological stimulation,and excessive secretion of extracellular matrix(ECM)with abnormal accumulation,is an important manifestation in the development of diabetic cardiomyopathy(DCM).Cardiac fibroblasts(CFs)are central to the pathogenesis of cardiac fibrosis in diabetes,and cardiac CFs can be activated to secrete large amounts of ECM under pathological conditions.DNA methylation is one of the most intensively studied major epigenetic modifications,occurring at gene promoters where DNA methylation often induces gene silencing and DNMT3 A is the key enzyme mediating DNA de novo methylation.A previous study confirmed that abnormalities in DNA methylation levels and elevated DNMT3 A expression exist during the development of fibrosis in diabetic myocardium.Methylenetetrahydrofolate reductase(MTHFR,5,10-methylenetetrahydrofolate reductase),whose main role is to convert5,10-methylenetetrahydrofolate to biologically active 5-methyltetrahydrofolate in the folate metabolic pathway,is a key enzyme in folate metabolism.A deficiency of MTHFR can induce several diseases including fibrogenesis and is closely related to cell proliferation,apoptosis,and other functions.It has been well-documented that MTHFR is regulated by DNA methylation.Pyroptosis,a new programmed cell death modality discovered and confirmed in recent years,is characterized as being dependent on inflammatory Caspase-1 and accompanied by the release of a large number of pro-inflammatory factors.Literature reported that the occurrence and development of DCM were accompanied by the occurrence of CFs pyroptosis,which further induced diabetic myocardial fibrosis.In this study,we used STZ intraperitoneally injection to induce a diabetic myocardial fibrosis model in mice,collected mouse myocardial tissue,and detected the expression changes of related genes and proteins in mice;Primary CFs were also extracted,and an in vitro cell model was established using high glucose stimulation to explore the molecular mechanism of DNMT3A-mediated MTHFR methylation and the correlation between MTHFR and pyroptosis of CFs in diabetic cardiac fibrosis.Based on the results of our previous study and our previous experiments,we applied multiple techniques to elucidate the molecular mechanism of DNMT3A-mediated MTHFR methylation-induced pyroptosis in CFs to promote myocardial fibrosis in diabetes.Objective: To investigate the mechanism of diabetic myocardial fibrosis and elucidate the molecular mechanism by which DNMT3 A mediates MTHFR methylation-induceded pyroptosis in CFs to promote myocardial fibrosis in diabetes.Methods : The diabetic myocardial fibrosis model in mice was induced by intraperitoneal injection of STZ into C57 mice and divided according to the experimental design.To verify the level of myocardial fibrosis by Sirius red staining and Masson staining,the expression of postn and MTHFR in the myocardium of STZ model mice should be detected by Western blot with immunofluorescence double staining.Primary CFs were extracted from newborn C57 mice and an in vitro cell model was established using high glucose stimulation,and cells were selectively silenced or overexpressed for DNMT3 A or MTHFR according to the experimental design,while changes in the methylation level of the MTHFR promoter were detected by BSP or MSP methods,changes in the binding level of DNMT3 A and MTHFR were detected by Ch IP methods,and changes in the pyroptosis level of CFs were detected by flow cytometry and scanning electron microscopy.Results: 1.MTHFR expression level changes in the myocardium of STZ model mice versus CFs under high glucose stimulationWestern blot coupled with immunofluorescence results demonstrated that MTHFR expression was decreased in the myocardium of STZ model mice compared with CFs under high glucose stimulation.2.Reduced expression of MTHFR is regulated by DNA methylationThe results of BSP and MSP proved that the methylation level of the MTHFR gene promoter was increased in STZ model mice and CFs stimulated by high glucose,and the results of the chip proved that the binding level of DNMT3 A to MTHFR was enhanced in the STZ model mice and CFs stimulated by high glucose.3.Mouse in vivo knockout of DNMT3 A decreases epigenetic repression of MTHFR Applying LV-DNMT3 A knockout mice DNMT3 A expression,Western blot with immunofluorescence results proved that in vivo DNMT3 A knockout mice alleviated the decrease of MTHFR induced by STZ modeling.And pathological staining with Western blot confirmed that knockdown of DNMT3 A in vivo alleviated STZ-induced diabetic myocardial fibrosis.BSP with MSP results demonstrated that the knockout of DNMT3 A in vivo decreased MTHFR gene promoter methylation levels induced by STZ modeling.4.Overexpression of MTHFR inhibits cardiac fibroblast pyroptosis under a high glucose environmentWestern blot coupled with flow cytometry results confirmed that pyroptosis markers were elevated in CFs stimulated with high glucose,and the results of flow cytometry,TUNEL assay,and SEM confirmed that after overexpression of MTHFR by OE-MTHFR plasmid application,CFs pyroptosis induced by high glucose stimulation was alleviated.5.Knockdown of DNMT3 A alleviates cardiac fibroblast pyroptosis induced by silencing of MTHFR under high glucose stimulationWestern blot coupled with flow cytometry results confirmed that silencing MTHFR induced pyroptosis in CFs stimulated with high glucose,while knockdown of DNMT3 A alleviated CFs pyroptosis induced by silencing of MTHFR under high glucose stimulation.Conclusions:1.DNMT3 A expression is elevated in diabetic cardiomyopathy along with reduced MTHFR expression.2.MTHFR expression is regulated by DNMT3A-mediated DNA methylation.3.Decreased MTHFR expression in diabetic cardiomyopathy induces pyroptosis in CFs.