Role of PNPT1 in cardiomyocyte apoptosis induced by oxygen-glucose deprivation / 南方医科大学学报

OBJECTIVE@#To explore the effect of inhibiting polyribonucleotide nucleotidyl-transferase 1 (PNPT1) on oxygen-glucose deprivation (OGD)-induced apoptosis of mouse atrial myocytes.@*METHODS@#Cultured mouse atrial myocytes (HL-1 cells) with or without OGD were transfected with PNPT1-siRNA or a negative control siRNA (NC-siRNA group), and the cell survival rate was detected using CCK-8 assay. The expression levels of ACTB and TUBA mRNA were detected with qPCR, and the protein expression of PNPT1 was detected with Western blotting. The apoptosis rate of the treated cells was determined with flow cytometry, the mitochondrial membrane potential was detected using JC-1 kit, and the mitochondrial morphology was observed using transmission electron microscope.@*RESULTS@#With the extension of OGD time, the protein expression levels of PNPT1 increased progressively in the cytoplasm of HL-1 cells (P < 0.05). Transfection with PNPT1-siRNA significantly reduced PNPT1 expression in HL-1 cells (P < 0.05). Exposure to OGD significantly enhanced degradation of ACTB and TUBA mRNA (P < 0.05) and markedly increased the apoptosis rate of HL-1 cells (P < 0.05), and these changes were significantly inhibited by transfection with PNPT1-siRNA (P < 0.05), which obviously increased mitochondrial membrane potential and improved mitochondrial morphology of HL-1 cells exposed to OGD.@*CONCLUSION@#Inhibition of PNPT1 improves mitochondrial damage and reduces degradation of apoptotic-associated mRNAs to alleviate OGD-induced apoptosis of mouse atrial myocyte.

The role of Tom70 in cardiac microvascular endothelial cell damage induced by high fat and high glucose / 解放军医学杂志

Objective To investigate the effect of high glycolipid on mouse cardiac microvascular endothelial cells (MCMECs),clarify the role of Tom70 (translocase of the outer mitochondrial membrane 70,Tom70) in it,and explore the related mechanism.Methods MCMECs cultured with normal glucose medium were divided into normal glucose group (NG,5.5mmol/L),high glucose group (HG,25mmol/L) and HG combined with high fat group (HG+HF,glucose concentration 25mmol/L,500μmol/L,16h).Then,the expression of Tom70 in MCMECs was knocked down by siRNA,and the HG+HF group was further divided into vehicle group,Scramble siRNA group and Tom70-siRNA group.To further confirm the specific mechanism of Tom70 in MCMEC injury induced by high glycolipid,Tom70-siRNA group was subgrouped into N-acetylcysteine (NAC)-free group and NAG-containing group.Accordingly,the apoptosis levels were measured by flow cytometer,the generation of nitric oxide (NO) was detected by ELISA kit,the expressions of Tom70 were determined by immunofluorescence and qRT-PCR,and the levels of reactive oxygen species (ROS) by DHE staining and ELISA kit.Results The apoptosis level increased after exposure to HG,and the generation of NO decreased (P＜0.05),while merging HF could aggravate these injuries (P＜0.05).Moreover,HG inhibited the expressions of Tom70 and promoted the production of ROS in MCMECs (P＜0.05).Compared with HG alone,and combination with HF significantly inhibited the expression of Tom70,and significantly increased the production of ROS (P＜0.05).Most importantly,compared with the vehicle group and Scramble siRNA group,the intracellular ROS content and apoptosis rate increased in the Tom70-siRNA group,while generation of NO was significantly decreased (P＜0.01).In contrast,these damage effects mentioned above were partially reversed by the application of antioxidants NAC (P＜0.05).Conclusions High fat can further aggravate the damage on diabetic MCMECs and Tom70 could exert its effect against cardiac microvascular endothelial injury induced by diabetes via inhibiting oxidative stress.

Inhibition of GPR35 attenuates ischemic myocardial injury / 解放军医学杂志

Objective To investigate the protective role of GPR35 inhibition on hypoxic myocardial cell line and mouse myocardial infarction (MI) model.Methods For investigating the changes of GPR35 expression in hypoxic environment,the murine myocardial cells (MCM) were divided into normoxia group and hypoxia group,the mRNA expression of GPR35 was determined by q-PCR and the protein level was measured by Western blotting 6h after incubation.For further studying the role of GPR35,MCM were divided into four groups:normoxia,hypoxia,hypoxia+vehicle,hypoxia+CID2745678 (GPR35 inhibitor,3μmol/L) group.Accordingly,the apoptosis of cardiomyocytes was measured by flow cytometer and TUNEL.For investigating the changes of GPR35 expression in the state of myocardial ischemia,the C57 male mice were divided into sham group (n=6) and MI group (n=8),the mRNA expression of GPR35 was determined by q-PCR and the protein level was measured by Western blotting 3 days after MI.For further studying the role of GPR35,the C57 mice were divided into four groups:sham (n=6),MI (n=8),MI+vehicle (n=8) and MI+CID2745678 (n=8) group.Ultrasound echocardiography was performed 4 weeks after MI.Mice were then sacrificed and the hearts were removed and stained with Masson to measure the myocardial fibrosis area.Results Compared with normoxia group,the levels of GPR35 mRNA and protein increased obviously in hypoxia group (P＜0.01,P＜0.05);Compared with hypoxia+vehicle group,the myocardial cells apoptosis in hypoxia+CID2745678 group decreased markedly (P＜0.05).Three days after MI,compared with the sham group,the levels of GPR35 mRNA and protein increased obviously (P＜0.01,P＜0.05) in MI group;Compared with MI+vehicle group,the left ventricular fraction shortening (LVFS) and left ventricular ejection fraction (LVEF) relieved obviously (P＜0.05) and myocardial fibrosis level declined markedly in MI+CID2745678 group (P＜0.05).Conclnsion Inhibition of GPR35 could decrease the apoptosis of cardiomyocytes cultured in hypoxia and attenuated the injury of myocardial ischemia.

Protection of MICU1 against myocardial hypertrophy induced by angiotensin Ⅱ / 解放军医学杂志

Objective To investigate the role of mitochondrial calcium uptake 1 (MICUI) in myocardial hypertrophy of mice and underlying mechanism.Methods The model of myocardial hypertrophy was established via incubation of mouse cardiac myocytes (MCM) with 300nmol/L angiotensin Ⅱ (Ang Ⅱ) for 48 hours in vitro.After that,MICU1 specific small interfering RNA (siRNA) was delivered to knockdown MICU1 levels in MCM.On the other hand,adenovirus-mediated over-expression of MICU 1 was transfected into MCM.Accordingly,the expressions of ANP and BNP in myocardial cells were measured by qRT-PCR.Mitochondrial membrane potential and ATP contents were detected byJC-1 assay kit and ATP assay kit,respectively.Then,Western blotting and qRT-PCR were used to detect the levels of MICU1 in myocardial cells.The mitochondrial Ca2+ contents were measured via atomic absorption flame spectroscopy.The size of myocardial cells was determined by α-actinin staining.Results Mitochondrial membrane potential and ATP contents in hypertrophic cardiomyocytes induced by Ang Ⅱ were both decreased.Meanwhile,myocardial hypertrophy significantly increased mitochondrial Ca2+ contents but decreased MICU1 levels.With the method of genetic intervention,we found that MICUI deficiency exacerbated mitochondrial Ca2+ overload,increased cell surface and elevated the expression of BNP.Conversely,the overexpression of MICU1 obviously decreased mitochondrial Ca2+ overload,cell surface of MCM and expressions of ANP and BNP.Conclusion MICU1 alleviates Ang Ⅱ-induced myocardial hypertrophy via inhibiting mitochondrial Ca2+ overload.

Effect of perilipin-5 on apoptosis of cardiac microvascular endothelial cells induced by high fat and high glucose in mice / 解放军医学杂志

Objective To investigate the effects and mechanisms of perilipin-5 (Plin5) on the apoptosis of mouse cardiac microvascular endothelial cells induced by high fat and high glucose.Methods The mouse cardiac microvascular endothelial cells (MCMECs) cultured with high glucose medium were respectively given 0,100,300 and 500μmol/L palmitic acid for 24 hours.In order to explore the effects and mechanisms of Plin5 on MCMECs injuries induced by high fat and high glucose,MCMECs exposed to 300μmol/L palmitic acid for 24 hours were divided into control group,Scra siRNA group and Plin5 siRNA group.The control group was only treated with transfection reagent,the Scra siRNA group was given treatment of transfection reagent and garbled RNA,the Plin5 siRNA group was given treatment of transfection reagent and Plin5 specific siRNA.In order to further confirm the specific mechanism of Plin5 in high fat/glucose inducing MCMECs injury,MCMECs in Plin5 siRNA group were divided into vehicle group and N-acetyl cysteine (NAC) group,and given the same intervention of high fat.The apoptotic rate was detected by flow cytometry,qRT-PCR and Western blotting were respectively used to detect the mRNA and protein expression of Plin5,and the intracellular reactive oxygen species (ROS) level was tested by DHE staining and ELISA kit.Results The apoptotic rate of MCMECs was increased in a fat concentration-dependent manner (P＜0.05).Compared with 0μmol/L palmitic acid group,the intracellular ROS content and the expression of Plin5 increased significantly in 300μmol/L palmitic acid group (P＜0.05).Compared with the control group and the Scra siRNA group,the intracellular ROS content and apoptotic rate increased significantly in Plin5 siRNA group under the action of 300μmol/L palmitic acid (P＜0.05).Compared with the vehicle group,the intracellular ROS content and apoptotic rate decreased remarkably in NAC group (P＜0.05).Conclusion With inhibition of oxidative stress,Plin5 may reduce the apoptosis of MCMECs induced by high fat and high glucose.