Effects Of Vitamin B6 On Cardiac Function In Mice Following Myocardial Infarction And The Underlying Mechanisms

BackgroundAcute myocardial infarction(AMI)is caused by acute occlusion of coronary arteries,which is characterized by suddenness,high mortality and poor prognosis.The reperfusion in ischemic area after infarction is closely related to the prognosis of patients,meanwhile,angiogenesis and formation of collateral circulation play an important role in the recovery after myocardial infarction.Upregulation of angiogenesis could improve the prognosis of patients.The migration,proliferation and tube formation of endothelial cells are vital to angiogenesis.Vascular endothelial growth factor(VEGF),fibroblast growth factor and angiopoietin can promote angiogenesis,which is regulated by various signal transduction pathways.It has been reported that AMPK activation can induce p38MAPK phosphorylation,increase VEGF mRNA stability and protein secretion,and thereby promoting angiogenesis.Also,another study reported that AMPK activation inhibits the necrosis of human umbilical vein endothelial cells(HUVECs)caused by oxygen glucose deprivation.Therefore,the activity of AMPK is closely related to endothelial cell function and angiogenesis.Vitamin B6(VitB6),also known as pyridoxine,is a water-soluble Vitamin.It is a coenzyme in human body and participates in a variety of metabolic reactions,such as gluconeogenesis and the elimination of reactive oxygen species.Some chronic diseases are caused by VitB6 deficiency.People who have VitB6 deficiency chronically have been reported to present higher incidence of cardiovascular events and worse prognosis,while,the underlying mechanism remains unknown.It has also been reported that low levels of circulating VitB6 are associated with increased levels of C-reactive protein(CRP),and CRP levels can reflect the prognosis of coronary artery disease(CAD)patients.Inflammatory reactions are usually over-activated in various cardiovascular events,and coincidentally,VitB6 enables inflammatory factors to be quickly cleared away.Besides,some scholars think that AMPK could suppress inflammation via AP-2?/kB?/NF-Kb pathway.Therefore,we hypothesized that adequate VitB6 supply could inhibit over-activated inflammation and increase angiogenesis by phosphorylating AMPK,thereby promoting cardiac function recovery after myocardial infarction(MI)and improving prognosis.Objectives1.To study whether VitB6 could activate AMPK.2.To investigate whether VitB6 could improve the function of HUVECs and increase angiogenesis in vitro and vivo.3.To verify whether VitB6 pre-treatment could ameliorate the cardiac function of post-MI mice.Methods1.HUVECs was stimulated by VitB6 in different concentration gradients and time gradients to determine the optimal treatment condition.2.The phosphorylation level of AMPK was detected in HUVECs treated with VitB6 by western blot.3.The wound scratch assay was used to observe the effect of VitB6 on the migration function of HUVECs.4.CCK-8 and MTT agents were used to detect the proliferation function of HUVECs treated with VitB6.5.The effect of VitB6 on angiogenesis was researched by tube formation assay in HUVECs.6.Compound.C,an inhibitor of AMPK,was used to block AMPK phosphorylation,and the effects of VitB6 on HUVECs were detect again,to verify whether AMPK is indispensable.7.Animal model8-week-old b129 mice were randomly divided into 2 groups.One group was given 2 mg/ml VitB6 drinking water,another was fed under normal conditions.After 14 weeks VitB6 administration,all mice experienced the ligation of left anterior descending coronary artery(LAD)following Erne Gao's protocol to make acute myocardial infarction.8.EchocardiographyThe cardiac function post-MI was assessed 2 weeks after surgery by echocardiography,the left ventricular diameter at end-systole or end-diastole(LVIDs or LVIDd),left ventricular ejection fraction(LVEF)and shortened fraction(FS)were calculated by the software of echocardiography.9.Histopathological stainingHE staining was used to assess the percentage of myocardial infarction area.The capillary density in ischemia area was detect by immunohistochemical staining of CD31 and a-SMA.10.The concentration of inflammatory factors in serum were evaluated by Luminex.11.The activity of AMPK and the level of angiogenesis biomarkers in ischemia tissue were measured WB.Results1.The optimal treatment condition of VitB6 in HUVECs was 0.5 mM and 24 hours,under which condition the phosphorylation level of AMPK was the highest.2.After 0.5 mM and 1.0 mM VitB6 stimulation for 24 hours,the migration capability of HUVECs was enhanced.3.While,no significant changes were found on the proliferation assay,although HUVECs were treated in same condition.4.Fortunately,the tubule formation ability of HUVECs was increased under the stimulation of 0.5 mM and 1.0 mM VitB6.5.The VEGFA in HUVECs was augmented by VitB6 management.6.After Compound.C pretreatment,the activity of AMPK and the increase of VEGFA was inhibited.7.The enhance of migration and tube formation in HUVECs stimulated by VitB6 disappeared because of the blocking of AMPK.8.The percentage of myocardial infarction region was reduced in mice with VitB6 drinking.9.The cardiac function of mice after myocardial infarction in VitB6 group were enhanced.The LVEF and FS increase,meanwhile,the LVIDd and LVIDs decrease.10.The density of capillary in the ischemia area in VitB6 group were incremental compared with the control group.11.The concentration of inflammatory factors such as TNF-a and IL-6 in serum of MI mice were higher than the sham group,and VitB6 could protect mice against the inflammation.12.The phosphorylation of AMPK and the level of VEGFA in ischemia tissue were higher due to the VitB6 treatment.Conclusion1.Adequate VitB6 supply can phosphorylate AMPK in vitro and vivo.2.VitB6 can improve the function of HUVECs via activing AMPK.3.Appropr:iate VitB6 intake can ameliorate cardiac function in mice post-MI.