Mechanisms Of The Energy Homeostasis Mediated By Vitamin D/VDR In Cardiomyocytes In Mice

Objective:Vitamin D is closely related to cardiovascular disease,inflammation,tumor and other diseases in addition to regulating calcium and phosphorus metabolism.Studies have linked vitamin D status to a range of metabolic diseases,including obesity,metabolic syndrome and type 2 diabetes.In vivo animal studies have shown that VDR receptor gene knockout(VDR^-/-)mice exhibit increased energy metabolic rate,increased oxidation capacity of fatty acids,and significantly reduced adipose tissue.There is increasing evidence that vitamin D deficiency plays an important role in the development of cardiovascular diseases.Vitamin D/vitamin D receptor(VD/VDR)lacks involvement in the pathogenesis of a variety of heart diseases,but the mechanisms are unclear.Heart energy metabolism,especially fatty acid oxidation,seems to be an important factor in the pathogenesis of cardiovascular disease.Energy substrate imbalance is one of the main causes of cardiac dysfunction.Sirtuin3 is an important member of the family of deacetylases in cardiac tissue,which regulates mitochondrial metabolism.It is a type of important post-translational modification,an important way to regulate enzyme activity to adapt to changes in cell metabolism and energy,and an important enzyme to regulate fatty acid oxidation.In the condition of high blood lipid,the activity of Sirt3 decreases,the acetylation level of mitochondrial protein significantly increases,and the acetylcoenzyme A level of the acetylase substrate in the mitochondria significantly increases,thus increasing the fatty acid-oxidation involved in the tricarboxylic acid cycle,resulting in excessive use of fatty acid as energy substrate,increased oxygen consumption and damage to cells.This study aims to investigate whether vitamin D regulates mitochondrial fatty acid oxidation through the Sirt3signaling pathway to protect cardiomyocytes.Methods:In this study,8-10 weeks of wild male ICR mice and CYP27B1^-/-gene knockout(KO)mice were used as the main research objects.Mice were randomly divided into wild control group(Ctrl),vitamin D intervention group(CCE),and KO group,with 10 mice in each group.The CCE group was given vitamin D drinking water(20 l choleverdin emulsion dissolved in 200 ml drinking water).The three groups were given normal diet and high-fat diet for 14 weeks.Body weight,rectal temperature and intake of food and water were recorded at 3pm(the lowest basal metabolic rate).The respiratory metabolic rate was monitored for 24 hours in a metabolic cage.Heart rate,blood pressure,electrocardiogram and myocardial contractility were recorded before sampling.Heart tissues and blood were collected from the mice.In addition,hl-1 cell lines of mouse myocardial cells were cultured in high-glucose DMEM medium and treated with active vitamin D(calcitrol)(final concentration 2 10-8 M/L)and oleic acid(OA),respectively.Changes in morphology and protein levels were observed after 24hours of culture.In order to observe the effect of VD on Sirt3,we constructed Sirt3 gene promoter into Pgl3 plasmid for the detection of luciferase reporter gene.In addition,exon fragments of Sirt3 gene were selected and constructed into the PX-458 plasmid of CRISPR/CAS9 to knock out Sirt3 gene in myocardial cells,so as to prove that VD/VDR regulates myocardial fatty acid metabolism through Sirt3 gene.Western blot was used to detect the expression of myocardial VDR,Sirt3,Lcad and mitochondrial complex.The m RNA of myocardium including VDR,Sirt3,Glut4 were detected by RT-PCR.Serum blood glucose,triglycerides,calcium ions,total cholesterol and free fatty acids were measured with the kit.Oil red O staining was used to detect the changes of lipid droplets in myocardium.Body composition analyzer was used to analyze the percentage of fat in body weight of mice in each group.The ability of Sirt3 promoter region to bind to VDR was detected by chromatin immunoprecipitation(Ch IP)method.In addition,to further verify the regulatory effect of VD on Sirt3,we established a kidney injury model to further observe the relationship between the VD and Sirt3.Results:1.Compared with the normal diet of wild mice and mice with vitamin D3(CCE)intervention and 1?(OH)ase^-/-mice,1?(OH)ase^-/-mice showed emaciation phenotype,lower body weight,lower body fat content and higher respiratory metabolic rate.In addition,1?(OH)ase^-/-mice had lower blood glucose levels,lower serum calcium,triglycerides,cholesterol,and free fatty acid levels.CCE mice did the opposite.2.The changes were observed after the high-fat diet of wild mice,CCE intervention mice and 1(OH)ase-/-mice.After the intervention of high-fat diet mice CCE,it was found that the CCE mice had low metabolism,high serum triglycerides and low free fatty acids,but their blood glucose was lower in the CCE group.3.after oil red O staining to detect myocardial tissue in mice found that CCE+HF group of myocardial lipid drops more than HF mice,but found that after detection of myocardial tissue protein and m RNA level of VD have no effect on the discrepancy on the free fatty acids in the myocardial cell membrane,changes occur in the mitochondrial membrane fatty acid intake on the sign of VD at the same time also increased the expression of glucose transporter protein.These results were also verified in vitro in cardiomyocyte lines.4.Left ventricular pressure-volume ring results showed that 1?(OH)ase^-/-mice showed abnormal myocardial function and decreased contractility.Direct detection of myocardial contractility in each group showed that the presence of lipid droplets in myocardial cells in the CCE+HF group did not affect myocardial function.On the contrary,data of myocardial contractility showed that CCE intervention alleviated the myocardial injury caused by high lipid.5.Sirt3expression was detected on the myocardial m RNA levels of wild mice,CCE intervention mice and 1?(OH)ase^-/-mice,and it was found that the lack of active vitamin D led to a significant decrease in Sirt3 expression,and the expression level of Sirt3 increased significantly after CCE supplementation.Sirt3 and its downstream gene Lcad showed an obvious change trend in the myocardium of mice induced by the high-fat diet.The high-fat state inhibited the expression of Sirt3,and the downstream target gene Lcad was activated and its expression was increased after excessive acetylation.After the administration of CCE and active vitamin D,Sirt3 expression increased,while Lcad expression decreased significantly.6.Fatty acid oxidation of the myocardium was completed in the mitochondria.The tem observation of the myocardium also showed that the myocardium was significantly damaged,the myocardium was disorganized and the mitochondria were swollen after hyperlipidemia.Further detection of myocardial cell proteins revealed increased expression of respiratory chain complexes that Shared metabolic substrates with Sirt3.7.The inhibitor of Sirt3,AGK7,was added to cell culture medium for cell treatment.Oil red staining showed no lipid droplets in the cells.Cell proteins were detected again,and it was found that protein levels did not change after inhibiting Sirt3,and VD/VDR no longer had a regulatory effect on its downstream Lcad and respiratory chain complexes.8.Changes in Sirt3 signaling pathway were detected after knockout of Sirt3 gene at the cell level,and cells treated with OA and 1,25(OH)2D3 were also given.Lcad and mitochondrial complex proteins downstream were not changed in each group.9.In myocardial tissue,VDR can bind VDRE on the promoter of Sirt3 gene,and VDR protein and Sirt3 gene can also interact directly.Through this direct regulation of transcriptional activity,the effect of VD/VDR on myocardial fatty acids is completed through the Sirt3 signal pathway.10.In renal injury caused by hyperuricemia,Sirt3 expression was low in the hyperuricemia group,and the expression of Sirt3 was significantly increased after CCE intervention.Conclusions:(1)VD/VDR upregulation of Sirt3 inhibits Lcad;(2)VD/VDR indirectly regulates mitochondrial complex through Sirt3;(3)VDR regulates its transcriptional activity by binding to the Sirt3 promoter VDRE;(4)VD/VDR inhibits excessive oxidation of mitochondrial lipids;(5)VD/VDR down-regulates lipid metabolism and up-regulates glucose intake to balance the energy homeostasis of cardiomyocytes;(6)VD regulates the expression of Sirt3 gene in mice model of hyperuricemia and renal injury.