| BackgroundDiabetic cardiomyopathy(DCM)can increase the risk of heart failure and death in diabetic patients.Though several studies have targeted DCM,no effective approaches are available to prevent its progression and development,and drugs aiming at improving glycemic control and cardiac dysfunction fail to reduce the incidence of DCM.Recently,abundant evidence reveals that there is a high prevalence of vitamin D deficiency in diabetic patients,and vitamin D deficiency is strongly associated with cardiovascular complications.In addition to the role of vitamin D in autoimmunity,infections,cancers and metabolic disease,studies have shown that vitamin D is also involved in autophagy.Autophagy plays an important role in the maintenance of normal heart function and morphology.Several studies have found the abnormal regulations of autophagy in both type 1 diabetes and type 2 diabetes.The canonical Wnt/?-catenin signaling is associated with the pathological processes of cardiac remodeling,myocardial hypertrophy and the interstitial fibrosis.Recent data also reveal that Wnt/?-catenin signaling is possibly implicated in the development of DCM.Besides,it has been demonstrated that ?-catenin and VDR can regulate mutually by the interaction between the C-terminus of ?-catenin and the activator function-2(AF-2)domain of VDR,and high levels of ?-catenin can promote the transcriptional activity of 1,25D3.Moreover,the activation of ?-catenin signaling can activate the mammalian target of rapamycin(mTOR)through the inhibition of GSK-3?.mTOR is involved in the regulation of cardiac function in adults and acts as a key component in pathways regulating autophagy.Aims(1)To investigate the myocardial hypertrophy and autophagy induced by high glucose in H9C2 cells and the effect of 1,25D3 treatment.(2)Type 1 diabetes model and lentivirus-mediated vitamin D receptor knock-down(Lenti-shVDR)model were established in male Sprague-Dawley rats,and the changes in cardiac function and the pathological abnormalities of hearts were measured;Rats were treated with 1,25D3 and /or VDR gene silencing and/or chloroquine(CQ)to clarify the effect of 1,25D3 on cardiac autophagy and cardiac structural and functional abnormalities.(3)In high-glucose cultured H9C2 cells and in type 1 diabetic rats,?-catenin/TCF4/GSK-3?/mTOR signaling pathway was investigated to determine whether this pathway was implicated in the mechanism by which 1,25D3 protects against DCM.Methods(1)In vitro,H9C2 cells were exposed to different concentrations of glucose(5.5,15,25,35 mM)or 1,25D3(25,50,100,200 and 400 nM).Autophagy and cell viability were measured and the optimal intervention concentrations of high glucose and 1,25D3 were selected;in high-glucose cultured H9C2 cells,1,25D3 and/or bafilomycin A1 were used to investigate the effect of 1,25D3 on autophagy;Western-blot analyses were adopted to measure the protein levels of VDR,LC3-II/I,P62 and Beclin1;immunofluorescence assay was used to measure the distribution of VDR in cells;transmission electron microscopy(TEM)analysis was used to determine the formation of autophagosomes.(2)Type 1 diabetes model was induced by intraperitoneal injection of STZ and randomized into 7 groups: control group(NG),diabetes(DM)group,diabetes+1,25D3 group(DM+VD3),diabetes+1,25D3+lenti-shVDR group(DM+VD3+shVDR),diabetes +lenti-shVDR group(DM+shVDR),diabetes+1,25D3+chloroquine group(DM+VD3+CQ),diabetes+ chloroquine group(DM+CQ).Blood pressure and echocardiography were measured after 8 weeks treatment with 1,25D3 and/or CQ;data on heart weight(HW),body weight(BW)and tibia length(TL)were collected;fasting blood glucose,serum insulin,serum cholesterol,triglyceride,creatinine and calcium were detected;serum and urine albumin,serum parathyroid hormone(PTH)and 25-hydroxy-vitamin D(25(OH)D)were measured by commercial ELISA kits;fluorescence microscope was used to observe the expression of green fluorescent protein(GFP)in the liver,kidney and heart tissues of rats transfected with lenti-shVDR;hematoxylin and eosin(HE)staining and Sirius-red staining were used to detect the myocardial histopathological changes;TEM analysis was used to detect the ultra-structural changes of hearts;immunohistochemistry assay were adopted to observe the expression levels of LC3 B and P62 in the left ventricular tissues;western-blot analyses were performed to measure the expression of VDR,LC3-II/I,P62,Beclin 1,?-catenin,TCF4,c-myc,GSK-3?/p-GSK-3?(Ser 9)and mTOR/p-mTOR at protein levels;qRT-PCR was used to detect the expression of VDR,?-MHC,ANF,BNP,?-catenin,TCF4 and c-myc at mRNA levels.(3)The high-glucose cultured H9C2 cells were treated with 1,25D3 and/or LiCl for 24 h,and cells were divided into six groups: normal control(NG,5.5mmol/L),NG+LiCl group,high glucose group(HG,25mmol/L),HG + LiCl group,HG+VD3 group and HG+VD3+ LiCl group;The qRT-PCR analyses were performed to detect the relative mRNA expressions of VDR,?-MHC,ANF,BNP,?-catenin,TCF4 and c-myc among different groups;western-blot analyses were used to detect the expression of VDR,LC3-II/I,P62,Beclin 1,?-catenin,TCF4,c-myc,GSK-3?,p-GSK-3?(Ser 9),mTOR and p-mTOR at protein levels;the distribution of ?-catenin was detected in H9C2 cells using immunofluorescence assay;the formation of autophagosomes among different groups was detected by TEM.Results(1)In H9C2 cells,western blot results revealed a significant decrease in LC3-II/I and Beclin-1 expression and an elevation in P62 levels in the presence of high glucose media(25 and 35mM)compared with 5.5 mM and 15 mM group;1,25D3 also increased the protein levels of LC3-II/I and Beclin1,and decreased the protein levels of P62 in a dose-dependent manner(no significant difference were observed at the concentrations of 100,200 and 400 nM);Besides,TEM analyses revealed more autophagic vacuoles in the presence of 1,25D3 at 100,200 and 400 nM;compared with HG+VD3 group,co-treatment with bafilomycin A1(Baf)increased the protein levels of LC3-II/I and P62,and the number of autophagosomes.(2)Compared with NG group,rats in STZ-treated groups had higher levels of blood glucose,and lower levels of serum insulin and body weight.Compared with NG and DM+VD3 group,rats in other groups exhibited higher concentrations of urine albumin and serum triglyceride.No significant differences were observed regarding serum albumin and TC concentrations among different groups.Besides,compared with NG group,serum 25(OH)D levels were decreased in DM and DM+CQ groups,while administration of 1,25D3 restored its concentrations in DM and CQ-treated group.Though silencing VDR increased serum 25(OH)D and PTH levels,no significant difference were observed concerning serum calcium levels among different groups.Moreover,rats among different groups had similar levels of heart rate and blood pressure.(3)Compared with NG group,rats in DM group showed higher values of HW/BW and HW/TL ratio.Compared with NG group,echocardiography revealed higher values of LVEDs/d diameter,LVIVs/d,LVPW thickness,and the EDV/ESV levels in diabetic rats,whereas EF%,FS% and the E/A ratio were reduced;HE and Sirius-Red staining showed the disorders in the myocardial cells and the increased levels of CVF% and PCA/LA ratio in DM group;further TEM analyses also showed disrupted myofibrils and the misalignment and aggregation of mitochondria.However,the myocardial abnormalities of diabetic rats were attenuated by 1,25D3 treatment for 8 weeks,but silencing VDR or co-treatment with CQ eliminated the protective effects of 1,25D3 on cardiac morphology and function in diabetic rats.(4)In STZ-induced diabetic rats,western blot analyses revealed a significant reduction of LC3-II/I and Beclin1 expression,and an increase of P62 levels in in DM group compared with the control.Besides,compared with DM group,treatment with 1,25D3 increased the protein levels of LC3-II/I and Beclin1,and decreased the protein levels of P62.Co-treatment with CQ and 1,25D3 additionally increased the protein levels of LC3-II/I and P62.However,silencing VDR reversed the effect of 1,25D3 on autophagy.(5)Compared with NG group,both the mRNA and protein expressions of ?-catenin,TCF4 and c-myc were significantly up-regulated in DM group.Besides,the protein levels of p-GSK-3?(Ser9)/GSK-3? and p-mTOR/mTOR were higher in DM group than that of the NG group.1,25D3 treatment decreased the expressions of ?-catenin,TCF4 and c-myc at protein and mRNA levels,and decreased the protein levels of p-GSK-3?(Ser9)/GSK-3? and p-mTOR/mTOR.However,silencing VDR in diabetic rats reversed this situation.(6)In high-glucose cultured H9C2 cells,compared NG group,cells in HG group showed higher expressions of ?-catenin,TCF4 and c-myc at mRNA and protein levels,and had higher protein levels of p-GSK-3?/GSK-3? and p-mTOR/ mTOR,this situation was reversed by 1,25D3 treatment.Besides,compared with HG+VD3 group,combination of 1,25D3 and LiCl inhibited autophagy and promoted cardiomyocyte hypertrophy,as demonstrated by the increased expression of ANF,BNP and ?-MHC mRNA levels and P62 protein levels,the decreased protein levels of LC3-II/I and beclin1,and the reduction of autophagic vacuoles.Conclusion(1)In H9C2 cells,high glucose promoted myocardial hypertrophyand inhibited myocardial autophagy,while treatment with 1,25D3reversed this situation.(2)In STZ-induced type 1 diabetic rats,cardiac autophagy was inhibited and diabetic rats exhibited impaired cardiac structure,the interstitial fibrosis and cardiac dysfunction;Co-treatment of 1,25D3 with VDR partly improved the impaired cardiac morphology and function by restoring autophagy in the hearts.(3)In high-glucose cultured H9C2 cells and in STZ-induced type 1 diabetic rats,the expression of ?-catenin/TCF4/GSK-3?/mTOR pathway were increased;In high-glucose cultured H9C2 cells,co-treatment of 1,25D3 with LiCl abolished the beneficial effect of 1,25D3 on autophagy,and caused myocardial hypertrophy.In conclusion,our findings suggest that the administration of 1,25D3 effectively improved cardiac dysfunction,myocardial hypertrophy and the interstitial fibrosis in diabetic rats through possible mechanisms associated with autophagy and the ?-catenin/TCF4/GSK-3?/mTOR signaling pathway. |
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