PM-induced Cardiac Lipid Deposition In Mice:Based On The Effect And Mechanism Mediated By FGFR1 Hypermethylation

Objective:Substantial evidence has shown that ambient PM exposure can increase the risk of heart-related disease morbidity and mortality,but its pathogenic mechanism is still unclear.Since DNA methylation impacts processes of heart disease,it might be useful to explore the potential mechanisms between PM exposure and heart disease.In this study,the epigenetic mechanism of PM-induced cardiotoxicity via FGFR1 hypermethylation was explored by constructing a real-ambient PM exposure model,a FGFR1 inhibition model in cardiomyocytes,a FGFR1 hyperexpression model in mouse heart tissue,and a cardiomyocyte methylation inhibition model.Methods:1.Construction of real-ambient PM exposed mouse modelFrom November 2018 to January 2019,C57BL/6 mice were exposed to PM for 6weeks in Shijiazhuang City,Hebei Province,using the real-ambient PM exposure system.The mice were randomly divided into Control and PM exposure group.Mice were placed in an independent ventilation cage and exposed to unfiltered and filtered outdoor air for 16 hours per day.1)The mice were sacrificed after PM exposure,TG,NEFA and ATP content detection kit were used to detect the content of TG,NEFA and ATP in mouse heart;q RT-PCR was used to detect the m RNA expression level of fatty acid metabolism-related genes,mitochondrial fission/fusion-related genes.2)FGFR1,DNMT1,DNMT3 A and DNMT3 B m RNA expression levels were detected by q RT-PCR;The expression of FGFR1 protein was detected by WB.BSP was used to detect the DNA methylation level of FGFR1 gene promoter region in mouse heart.2.Construction of AC16 recovery model after PM exposureAC16 were treated with PM for 24 and 48 hours,respectively,and the PM was washed off at the end of exposure,and AC16 were cultured normally for 3 and 6 days to construct a cell recovery model.The DNA methylation level of FGFR1 gene promoter region in AC16 were detected by MSP.3.Construction of FGFR1 expression inhibition model in cardiomyocytesAC16 cardiomyocytes were treated with FGFR1 inhibitor(PD173074)to construct an FGFR1 inhibition model.The experiments were divided into 4 groups: Control,PM,PD173074,and PM + PD173074.After the treatment,WGA staining was used to detect the morphology of cardiomyocytes.The lipid accumulation was detected by Nile red staining.The mitochondrial content and membrane potential of cardiomyocytes were detected by Mito-green staining and JC-1 staining,respectively.q RT-PCR and WB were used to detect the m RNA and protein levels of AMPK/PGC1α pathway-related genes.4.Construction of methylation inhibition model in cardiomyocytesAC16 cardiomyocytes were treated with a methylation inhibitor(5-AZA)to construct a methylation inhibition model.The experiment was divided into 4 groups: Control,PM,5-AZA,and PM + 5-AZA.After the treatment,WGA staining was used to detect the morphology of cardiomyocytes.Nile red staining was used to detect lipid accumulation.Mito-green staining and JC-1 staining were used to detect mitochondrial content and membrane potential in cardiomyocytes,respectively.q RT-PCR and WB were used to detect the m RNA and protein levels of AMPK/PGC1α pathway-related genes.5.Construction of mouse heart FGFR1 overexpression model and real-ambient PM exposureThe mouse cardiac tissue FGFR1 overexpression model was established by tail vein injection of AAV9.From November 2022 to March 2023,C57BL/6 mice were exposed to PM for 15 weeks in Shijiazhuang City,Hebei Province,using the real-ambient PM exposure system.The cells were divided into 6 groups: Control,PM,AAV-CTRL,AAVCTRL+PM,AAV-FGFR1,AAV-FGFR1+PM.Cardiac function was detected by echocardiography.Lipid metabolism in the heart of mice was detected by Oil red O staining,TG,NEFA and ATP detection kits.q RT-PCR and WB were used to detect the m RNA and protein levels of AMPK/PGC1α pathway-related genes.q RT-PCR and WB were used to detect the m RNA and protein levels of AMPK/PGC1α.Results:1.Real-ambient PM exposure causes cardiac lipid deposition and abnormal lipid metabolism in miceCompared with the Control group,the levels of TG and NEFA in the hearts of PM exposed mice were increased,and the level of ATP was decreased.The m RNA levels of lipid metabolism-related genes including CD36,FABP3 and CPT1 B were decreased.The m RNA levels of mitochondrial fission/fusion related genes including MFN1,MFN2 and OPA1 were decreased.2.Real-ambient PM exposure causes elevated levels of DNMTs and FGFR1 gene methylation in mouse heartsWB and q RT-PCR showed that FGFR1 expression level was decreased and DNMT1,DNMT3 A and DNMT3 B levels were increased by PM exposure compared with the Control group.BSP analysis showed that PM exposure resulted in increased methylation levels in the promoter region of the FGFR1 gene.3.Change in FGFR1 methylation in cardiomyocytes after removal of PM exposureMSP showed that PM-exposed cardiomyocytes,after PM removal and a period of recovery,remained hypermethylated in the promoter region of the FGFR1 gene.4.Abnormal effects of FGFR1 inhibition on mitochondria,lipid deposition and AMPK/PGC1α pathway in cardiomyocytes1)Compared with the Control group,cardiomyocytes in the PD173074 and PM +PD173074 groups showed hypertrophy,lipid accumulation,reduced mitochondrial number and decreased mitochondrial membrane potential.This was similar to the results of the PM group.2)WB and q RT-PCR showed that PD173074,PM + PD173074 and PM treatment all resulted in decreased m RNA and protein levels of AMPK/PGC1α.5.Effect of methylation inhibition on DNMTs and FGFR1 gene expression in cardiomyocytesq RT-PCR showed that the levels of DNMT1,DNMT3 A and DNMT3 B were decreased in AC16 cells treated with 5-AZA compared with the Control group.5-AZA treatment ameliorated the decrease in FGFR1 gene expression in cardiomyocytes induced by PM exposure.6.Effects of methylation inhibition on mitochondria,lipid deposition and AMPK/PGC1α pathway in cardiomyocytesAfter 5-AZA treatment,myocardial hypertrophy,lipid accumulation,reduced mitochondrial number and decreased mitochondrial membrane potential induced by PM exposure were significantly improved.7.Alterations in cardiac function in FGFR1 overexpressing mice exposed to atmospheric PMEchocardiography showed that LVEDV,LVESV,LVIDd,LVIDs,SV and CO increased in the PM group compared with the Control group,and all indexes in the AAVFGFR1+PM group showed a downward trend compared to the PM exposure group,which was close to the cardiac function indexes of the Control group.There was no significant difference in cardiac function between the AAC-CTRL and the Control groups,and between the AAC-CTRL + PM and the PM groups.8.Alterations in cardiac lipid metabolism and AMPK/PGC1α pathway in cardiac FGFR1 high expressing mice exposed to atmospheric PMOil red O staining,TG,NEFA and ATP detection results showed that FGFR1 overexpression significantly improved cardiac lipid metabolism disorders induced by PM exposure.WB and q RT-PCR analysis showed that FGFR1 overexpression reversed the decrease of AMPK/PGC1α m RNA and protein levels caused by PM exposure.Conclusion:1.Real-ambient PM exposure induced mouse cardiac lipid deposition.2.PM exposure induced FGFR1 hypermethylation and inhibited its expression.3.FGFR1 regulates cardiac lipid levels and mitochondrial biogenesis via AMPK/PGC1α signaling pathway.4.FGFR1 overexpression alleviated PM exposure-induced cardiac dysfunction and lipid deposition.