Vitamin D Regulates Mitochondria-associated Endoplasmic Reticulum Membranes To Improve Diabetic Cardiomyopathy By The VDR/Pin1 Pathway

Objective:In this study,the model of diabetic SD rat induced by streptozotocin（STZ）was conducted to observe the protective effect of vitamin D（VitD）on the structure and function of heart,the expression of peptidyl-prolyl cis/trans isomerases 1（Pin1）,AMP-dependent protein kinaseα2（Ampkα2）,FUN14 domain-containing protein 1（Fundc1）,inositol 1,4,5-triphosphate type 2 receptor（Ip3r2）,and the formation of mitochondria-associated endoplasmic reticulum membranes（MAM）in left ventricular tissue;cell experiment was conducted to observe the protective effect of VitD on the apoptosis of neonatal rat cardiomyocytes（NRCMS）induced by high glucose and its influence on the MAM formation and mitochondrial function regulated by Pin1,and to explore the possible regulatory mechanism of vitamin D in the diabetic cardiomyopathy.Method:Animal experiment:SD rats were intraperitoneally injected with STZ to construct a diabetic cardiomyopathy model.Animal groups:Control group（normal control）,Control+Cal group（normal control+calcitriol 1μg/kg/d）,DM group（STZ-SD rats）,DM+Cal group（STZ-SD rats+calcitriol 1μg/kg/d）,10 rats in each group.Fasting blood glucose was determined by the glucose oxidase method;serum biochemical parameters were measured by the automatic biochemical analyzer;serum concentrations of ANP,BNP and VitD were examined by ELISA;cardiac structure and function in SD rats were evaluated by echocardiogram;myocardial collagen deposition of SD rats were assessed by Sirius red（SR）staining;apoptosis in myocardium of the rats were assessed by TUNEL staining;the amount of MAM were measured by transmission electron microscope in rat myocardium;the expression levels of VDR,Pin1,Ampkα2,pAmpk,Fundc1,Ip3r2,Bax and Bcl2 proteins in left ventricle were examined by Western blotting.Cell experiment:NRCMS were isolated and cultured by differential adherence method,and identified through morphology and immunofluorescence.A high glucose-induced cardiomyocyte injury model was established by treating with 33 m M glucose;cell viability of NRCMS were measured by CCK8 experiment;mitochondrial reactive oxygen species（MitoROS）,mitochondrial calcium ions([Ca²⁺]^m),and mitochondrial membrane potential（MMP）in NRCMS were measured by flow cytometry.The formation of MAM in NRCMS were observed by laser confocal microscope;the protein levels of VDR cytoplasm/nucleus distribution,Pin1,Ampkα2,pAmpk,Fundc1 and Ip3r2 were measured by Western blotting.The expression of Pin1 and VDR were silenced by transfection of siRNA,and the effect of Ip3r2 changes mediated by Pin1 and VDR siRNA on the MAM and mitochondrial function of NRCMS were observed.To observe whether VDR affected the expression of Pin1 at the transcription level,the Pin1 luciferase reporter plasmid and the VDR overexpression plasmid were constructed and co-transfected into 293T cells,the cells were lysed after 24 h for luciferase analysis by chemiluminescence.Result:Animal experiment:1)Compared with control group,the fasting blood glucose,serum TC,LDL-c and TG levels of DM group were significantly increased（p<0.05）,the serum 1,25-Dihydroxyvitamin D3（DHVD3）level was significantly reduced（p<0.05）;compared with DM group,there were no significant change in the fasting blood glucose,serum TC,LDL-c,TG and DHVD3 levels in DM+Cal group（p>0.05）;there were no significant difference in the serum HDL-c,calcium,phosphorus,ANP and BNP levels of rats in each group;2)The echocardiogram showed that compared with control group,the EF,FS and E/A in diabetic rats were significantly reduced（p<0.05）,and the EDV,ESV,LVEDD and LVESD were significantly increased（p<0.05）;compared with DM group,the EF,FS and E/A of rats in DM+Cal group were significantly improved（p<0.05）;3)TUNEL test showed that compared with control group,the apoptosis of cardiomyocytes in DM group was significantly increased（p<0.05）;compared with DM group,the apoptosis of cardiomyocytes in DM+Cal group was significantly reduced（p<0.05）;4)Sirius red staining showed that there was only a little filamentous red staining in the myocardial fiber septum of the rats in control group and control+Cal group;a large number of reticular and flaky red stains was seen in the myocardial fiber septum of rats in DM group;compared with DM group,the myocardial collagen volume fraction in DM+Cal group was significantly reduced（p<0.05）;5)Transmission electron microscopy showed that compared with control group,the formation of MAM in DM group significantly increased（p<0.05）;compared with DM group,the number of MAM in DM+Cal group was significantly decreased（p<0.05）;6)Compared with control group,the protein levels of Pin1,Fundc1,Ip3r2 and Bax in the myocardial tissue of rats in DM group were significantly increased（p<0.05）,the protein levels of VDR,pAmpk and Bcl2 were significantly reduced（p<0.05）;compared with DM group,the protein levels of Pin1,Fundc1,Ip3r2and Bax in the myocardial tissue of rats in DM+Cal group were significantly decreased（p<0.05）,the protein levels of VDR,pAmpk and Bcl2 were increased（p<0.05）.Cell experiment:1)Compared with NG group,the protein levels of VDR and pAmpk of NRCMS in HG group were significantly reduced（p<0.05）,and the protein levels of Pin1,Fundc1 and Ip3r2 were significantly increased（p<0.05）;compared with HG group,the protein levels of VDR and pAmpk of NRCMS down-regulated by high glucose were significantly increased after VitD treatment（p<0.05）,and the protein levels of Pin1,Fundc1,Ip3r2 were significantly decreased（p<0.05）;2)Compared with NG group,the formation of MAM,mitochondrial calcium and reactive oxygen species of NRCMS in HG group were significantly increased,the mitochondrial membrane potential was significantly reduced,and the cell apoptosis rate was increased（p<0.05）;compared with HG group,the formation of MAM,mitochondrial calcium and reactive oxygen species of NRCMS induced by high glucose were significantly reduced after VitD treatment,the mitochondrial membrane potential was significantly improved,and the cell apoptosis rate was reduced（p<0.05）;3)Down-regulating the expression of VDR by transfection of VDR siRNA,the inhibitory effect of VitD on HG-induced MAM formation and mitochondrial dysfunction were weakened;down-regulating the expression of Pin1 by transfection of Pin1 siRNA,which could alleviate HG-induced MAM formation and mitochondrial dysfunction;4)293T cells were co-transfected with Pin1 luciferase reporter plasmid and VDR overexpression plasmid,luciferase analysis showed that overexpression of VDR inhibited the transcription level of Pin1.Conclusion:1)Vitamin D could regulate the abnormal expression of pAmpk,Fundc1,Ip3r2 protein and the formation of MAM,and improve the cardiac structure and function of diabetic rats;2)Activating the VDR or inhibiting the Pin1 could reduce the formation of MAM in NRCMS by up-regulating the phosphorylation level of Ampk,and improve the function of myocardial mitochondria;3)Vitamin D regulated the expression of Pin1 at the transcription level through VDR,thereby affecting the formation of MAM and improving diabetic cardiomyopathy.