Trimethylamine-N-oxide Promotes The Development And Progression Of Calcific Aortic Valve Disease Via Regulation Of Endoplasmic Reticulum-mitochondrial Stress

Background:The choline-derived metabolite trimethylamine N-oxide(TMAO),a gut microbiota-generated metabolite,is associated with cardiovascular diseases.TMAO promotes cardiovascular disease through the induction of inflammation and stress.Inflammatory responses and stress are involved in the progression of calcified aortic valve disease(CAVD).However,whether TMAO mediates CAVD remains unclear.Objective:In vitro expriments and mice models will be performed to investigate the relationship between TMAO and CAVD.We hypothesize that TMAO promotes the progression of CAVD via modulating endoplasmic reticulum(ER)-mitochondrial stress.This would provide a new sight in the pathogenesis of CAVD and attractive potential targets for the prevention,diagnosis and treatment of CAVD.Methods:Human studies:Patients with/without aortic valve calcification according to echocardiography were recruited.Fasting blood samples were collected and plasma samples were measured by HPLC/MS/MS immediately after preparation.In vitro:(1)AVICs were isolated and stimulated by TMAO,Reactive oxygen species,mitochondrial membrane potential were detected.The levels of ALP,BMP-2,ATF-4,XBP-1s,CHOP and IRE-1α phosphorylation were detected by Western blot.The mitochondrial and endoplasmic reticulum(ER)morphology were observed by transmission electron microscopy(TEM).Calcium deposits were detected by Alizarin red S staining.(2)Cells were treated with tauroursodeoxycholic acid(TUDCA),N-acetyl-L-cysteine(NAC),IKK inhibitor(Bay11-7082),ATF-4 siRNA and XBP-1 siRNA prior to stimulation with TMAO.The levels of ALP and BMP-2 were detected by Western blot.Calcium deposits were detected by Alizarin red S staining.In vivo:Adult(12-14 months old)C57BL/6 male mice were randomly,assigned to groups fed a regular diet(CTRL),high choline diet(HCD),high fat diet(HFD),high fat and choline diet(HFCD)or HFCD with 3,3-Dimethyl-1-butanol(DMB,1.0%)in the drinking water.Mice were housed and observed for 16 weeks.Blood was obtained for the measurement of TMAO levels.The hearts were prepared for HE and immunohistochemistry staining.Results:1.To observe the association between plasma TMAO levels and the incidence risk of CAVD:the plasma TMAO levels were significantly higher in the CAVD group(2.87μmol/l)than in the non-CAVD group(1.90μmol/l).Multivariate logistic regression analysis showed that TMAO was significantly correlated with CAVD.2.To investigate the association between TMAO and osteogenic responses in human AVICs:stimulation with TMAO resulted in a dose dependent increase in the protein levels of BMP-2 and ALP.In addition,TMAO promoted the accumulation of calcium deposits and the formation of calcification nodules in human AVIC cultures.3.To analyse the effect of TMAO.on ER and mitochondrial stress signals in human AVICs:TMAO induced the phosphorylation of IRE-1α and increased the protein levels of ATF-4,XBP-1s,and CHOP.TMAO treatment increased intracellular ROS levels and decreased MMP in human AVICs.The electron microscopy results revealed that TMAO treatment enlarged the morphology of the mitochondria and ER.4.To determine whether ER stress and mitochondrial stress pathways both involved in TMAO-induced osteogenic responses in human AVICs:TUDCA and NAC treatments markedly inhibited the phosphorylation of IRE-1α and decreased the protein levels of ATF-4,XBP-1s,and CHOP.Pretreatment with TUDCA and NAC also attenuated TMAO-induce ROS production and reversed distorted mitochondria and dilated ER.Treatment with TUDCA and NAC resulted in an increased reduction in the levels of BMP-2 and ALP and the formation of calcium deposits in human AVICs.Silencing of both ATF-4 and XBP-1 also reduced the protein levels of BMP-2 and ALP following TMAO simulation.5.To examine whether NF-κB involved in TMAO-induced ER stress and mitochondrial stress pathways to increase osteogenic responses in human AVICs:TMAO induced the phosphorylation of NF-κB.TUDCA and NAC treatments markedly inhibited the phosphorylation of NF-κB.The inhibition of NF-κB with Bay11-7082 reduced the levels of BMP-2 and ALP and calcium deposit formation.6.To determine the effect of different diets on serum TMAO and aortic valve thicknesses in mice:the serum TMAO levels,aortic valve thicknesses and the levels of BMP-2 in the valvular tissue in mice fed HCD,HFD and HFCD were increased compared with those in mice fed a regular diet.Similar observations were made in the HFCD group compared with the HFD group.However,compared with the HCD mice,the HFD mice displayed accelerated aortic valve thickening but lower serum TMAO.HFCD mice orally administered DMB showed a significant reduction in serum TMAO levels and aortic valve thickness.Conclusion:1.Patients with CAVD have increased levels of plasma TMAO.TMAO is significantly correlated with CAVD.2.TMAO induces osteogenic responses in human AVICs.3.TMAO induces ER stress and mitochondrial stress in human AVICs.4.TMAO induces osteogenic responses via ER-mitochondrial stress in human AVICs.5.TMAO induces osteogenic responses through ER-mitochondrial stress-induced NF-κB activation in human AVICs.6.Supplemental choline or fat diet elevates circulating TMAO levels and increases aortic valve lesions in vivo.Aortic valve leaflet thickness is reversed by treatment with DMB,which inhibited the choline-induced increase in plasma TMAO levels.