Role of Drp1 in high glucose-induced rat cardiomyocyte hypertrophy

AIM: To investigate the effect of dynamin-related protein 1 (Drp1) on high glucose-induced cardiomyocyte hypertrophy. METHODS: The primarily cultured rat cardiomyocytes were divided into normal glucose group, mannitol group, DMSO group, high glucose group and high glucose+mitochondrial division inhibitor-1 (Mdivi-1) group. The area of single cardiomyocyte was calculated by wheat germ agglutinin staining. The mitochondrial membrane potential of the cardiomyocytes was detected by JC-1 staining, and the expression levels of atrial natriuretic peptide (ANP), the marker of cardiac hypertrophy, Drp1 and its phosphorylated proteins p-Drp1 (Ser616) and p-Drp1 (Ser637) were determined by Western blot. RESULTS: Compared with normal glucose group and mannitol group, the cell area was increased significantly and the mitochondrial membrane potential was decreased significantly in high glucose group. The protein levels of ANP and p-Drp1 (Ser616) were significantly increased, the protein level of p-Drp1 (Ser637) was decreased, and no significant difference of total Drp1 level among groups was observed. In high glucose+Mdivi-1 group, ANP expression and the protein level of p-Drp1 (Ser616) were significantly decreased, and the protein level of p-Drp1 (Ser637) was increased as compared with high glucose group. CONCLUSION: High glucose induces hypertrophy of cardiomyocytes, and the mechanism is associated with increased mitochondrial fission due to changes of Drp1 phosphorylation levels.     

Role of ubiquitin E3 ligase TRIM10 in regulating cardiomyocyte hypertrophy

AIM: To explore the role of ubiquitin E3 ligase tripartite motif 10 (TRIM10) in the development of cardiomyocyte hypertrophy. METHODS: Primary cultured neonatal rat cardiomyocytes (NRCMs) were infected with siRNA-TRIM10, siRNA-control, Ad-TRIM10 or Ad-GFP for 24 h respectively, and then stimulated with phenylephrine (PE) for additional 24 h. The protein levels of TRIM10, AKT and ERK1/2 were determined by Western blot. The size of the NRCMs was measured by immunofluorescence staining. The mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was detected by RT-qPCR. RESULTS: Compared with the control, PE treatment significantly increased the protein expression of TRIM10. Moreover, transfection of NRCMs with siRNA-TRIM10 markedly inhibited cardiomyocyte size, the mRNA expression of ANP and BNP, and the phosphorylation levels of AKT and ERK as compared with siRNA-control after PE treatment. In contrast, overexpression of TRIM10 significantly enhanced PE-induced hypertrophic effect on NRCMs above. CONCLUSION: TRIM10 regulates cardiomyocyte hypertrophy partially through AKT and ERK signaling pathways.     

Activation of TGR5 reduces high glucose-induced cardiomyocyte hypertrophy by inhibiting CaN/NFAT3 signaling

AIM: To investigate the role of G-protein-coupled bile acid receptor 1(GPBR1; also known as TGR5) activation in high glucose-induced cardiomyocyte hypertrophy and calcineurin (CaN)/nuclear factor of activated T-cells 3 (NFAT3) signaling.METHODS: Primarily cultured mouse cardiomyocytes were used in the study. The cell surface areas of the cardiomyocytes were measured by an image analysis system. The cell protein content was detected by BCA method. The expression of TGR5, CaN and NFAT3 at mRNA and protein levels was determined by RT-PCR and Western blot.RESULTS: The mouse cardiomyocytes were successfully cultured. High glucose significantly induced the increases in the cell surface area, the cell protein content and the expression of CaN and NFAT3 (P<0.05) in the cardiomyocytes. TGR5 activation or a CaN antagonist cyclosporin A inhibited high glucose-induced cardiomyocyte hypertrophy and the expression of CaN and NFAT3 (P<0.05). These effects of TGR5 activation were abolished by TGR5 gene interference (P<0.05).CONCLUSION: TGR5 activation reduces high glucose-induced cardiomyocyte hypertrophy by inhibiting CaN/NFAT3 signaling.     

Over-expression of SIRT1 gene inhibits high glucose-stimulated H9c2 cardiomyocyte apoptosis and ROS level through PI3K/AKT signaling pathway

AIM: To investigate the effects of silent information regulator 1 (SIRT1) over-expression on the apoptosis and the level of reactive oxygen species (ROS) in high glucose-induced H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were transfected with empty plasmid (pcDNA3.1-NC) and SIRT1 over-expression plasmid (pcDNA3.1-SIRT1), and then stimulated by high glucose. The H9c2 cells were divided into control group, high glucose group, high glucose + pcDNA3.1-NC group and high glucose + pcDNA3.1-SIRT1 group. The expression of SIRT1 at mRNA and protein levels in each group was determined by qPCR and Western blot. The viability of the cells was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The protein levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, AKT and phosphorylated AKT were examined by Western blot. RESULTS: SIRT1 was significantly decreased in high glucose-induced H9c2 cardiomyocytes, the cell viability was significantly decreased compared with control group, while the ROS levels and apoptotic rate were increased, and the phosphorylated PI3K and AKT protein levels were down-regulated (P<0.05). Over-expression of SIRT1 significantly promoted the viability of H9c2 cardiomyocytes induced by high glucose, decreased the ROS levels and apoptotic rate, and up-regulated phosphorylated PI3K and AKT protein levels (P<0.05). CONCLUSION: SIRT1 over-expression reverses the decrease in the viability of high glucose-stimulated H9c2 cardiomyocytes, and the increases in apoptotic rate and oxidative stress by regulating PI3K/AKT signaling pathway.     

PPAR-α involves in cardiomyocyte hypertrophy induced by high glucose and insulin

AIM: To study the role of peroxisome proliferator-activated receptor-α (PPAR-α) signal transduction pathway in cardiac hypertrophy induced by high glucose and insulin (HGI). METHODS: The cultured neonatal rat cardiomyocytes were used to observe the effect of fenofibrate (FF), a selective PPAR-α agonist, on cardiomyocyte hypertrophy induced by HGI (glucose at concentration of 25.5 mmol/L and insulin at 0.1 μmol/L). The cardiomyocyte hypertrophic responses were assayed by measuring the cell surface area, protein content, and mRNA expression of atrial natriuretic factor (ANF). The expressions of mRNA and protein were assayed by real -time PCR and Western blotting. RESULTS: In cultured cardiomyocytes, HGI induced profound change of hypertrophic morphology, the significant increase in cell surface area, protein content and ANF mRNA expression compared to those in vehicle control (P<0.01), but the expressions of PPAR-α mRNA and protein decreased significantly (P<0.05). At the same time, the expression of cyclooxygenase 2 (COX-2), one of the PPAR-α downstream effectors was obviously elevated (P<0.05). However, FF (0.1, 0.3 and 1 μmol/L) inhibited the cardiomyocyte hypertrophy induced by HGI in a concentration-dependent manner (P<0.01). FF at concentration of 0.3 μmol/L increased the expressions of PPAR-α in both mRNA and protein levels (P<0.05) and inhibited the expressions of COX-2 (P<0.05), which were abolished by MK 886 (0.3 μmol/L), a selective PPAR-α antagonist (P<0.05). CONCLUSION: PPAR-α signal transduction pathway and its downstream effector COX-2 might involve in the cardiomyocyte hypertrophy induced by HGI.     

Role of TRIM8 in mouse cardiomyocyte apoptosis induced by high glucose and high free fatty acid

AIM: To investigate the effects of tripartite motif-containing protein 8 (TRIM8) on the apoptosis of mouse cardiomyocytes (MCMs) induced by high glucose and high free fatty acid (HGHF) and the underlying mechanism. METHODS: The MCMs were divided into normal glucose (NG) group (glucose at 5.5 mmol/L), high glucose (HG) group (glucose at 33 mmol/L), high free fatty acid (HF) group (sodium palmitate at 300 μmol/L) and HGHF group (glucose at 33 mmol/L and sodium palmitate at 300 μmol/L). The expression of TRIM8 in the MCMs was knocked down by siRNA, and the MCMs was further divided into control group, scrambled siRNA (Scra-siRNA)/PBS group, TRIM8-siRNA/PBS group, Scra-siRNA/HGHF group and TRIM8-siRNA/HGHF group. To further confirm the specific mechanism of TRIM8 in the MCM injury induced by HGHF, the MCMs were subgrouped into HGHF/DMSO group, HGHF+TRIM8-siRNA+DMSO (HGHF+Ts/DMSO) group, HGHF/ML385 group and HGHF+Ts/ML385 group. Accordingly, apoptosis was analyzed by flow cytometry, and the levels of reactive oxygen species (ROS) were measured by flow cytometry and DHE staining. The expression of TRIM8, nuclear factor E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1) at mRNA and protein levels was determined by qPCR and Western blot. RESULTS: HGHF increased the expression of TRIM8, and suppressed the expression of Nrf2, GCLC, HO-1 and NQO-1 in the MCMs (P < 0.05). Compared with Scra-siRNA/HGHF group, the intracellular ROS content and apoptotic rate were decreased in TRIM8-siRNA/HGHF group (P < 0.05). Correspondingly, the expression of the antioxidant molecule Nrf2 and its downstream genes GCLC, HO-1 and NQO-1 was increased (P < 0.05). In contrast, the addition of Nrf2 inhibitor ML385 partially reversed the inhibitory effect of TRIM8 expression knock-down on HGHF-induced apoptosis of MCMs. CONCLUSION: TRIM8 exacerbates the HGHF-induced cardiomyocyte apoptosis by modulating Nrf2 antioxidative pathway.     

Inhibition of Rac1 protects cardiac functions in STZ-induced diabetic cardiomyopathy through decreasing pho-p38 MAPK

AIM: To investigate the effects of Rac1 inhibition on the size of cardiomyocytes and left ventricular functions in diabetic cardiomyopathy. METHODS: Type 1 diabetes was induced in 8-week old C57 mice by streptozotocin (STZ, ip injection). Diabetic mice were treated with NSC23766, a specific inhibitor of Rac1 (STZ+NSC group, n=15) or without treatment (STZ group, n=15). Nondiabetic mice were used to serve as empty control (Con group, n=10) or NSC23766 control (NSC group, n=10). The survival rate, LVHW/BW and left ventricular functions were detected at the end of 8 weeks after induction of diabetes. The expression of ANP, BNP, β-MHC and pho-p38 MAPK in the cardiac tissues were analyzed by real-time RT-PCR and Western blotting, respectively. Hematoxylin and Eosin staining (HE) was used to measure the cell size in the cardiac tissues. RESULTS: The functions of left ventricle were significantly impaired in the diabetic animals with decreased survival rate and increased LVHW/BW, which was accompanied by significant increases in ANP, BNP and β-MHC, and elevated pho-p38 MAPK expression in diabetic hearts. The increased survival rate, improved left ventricular functions and decreased LVHW/BW were observed in the diabetic animals treated with NSC. The mRNA expression of ANP, BNP, β-MHC and pho-p38 MAPK was significantly attenuated in the diabetic hearts by NSC treatment. The size of the cardiomyocytes, which increased in diabetic hearts, was decreased in the NSC-treated diabetic cardiac tissue. CONCLUSION: Rac1 inhibition protects left ventricular functions and attenuates hypertrophy, which is associated with the decrease in pho-p38 MAPK expression in diabetic heart. These data suggest that inhibition of Rac1 might be beneficial to diabetic cardiomyopathy.     

Regulation of AMPK/PPARα/SCAD signal pathway on cardiac hypertrophy

AIM：To study the effect of short-chain acyl-coenzyme A dehydrogenase (SCAD）on cardiac hypertrophy and to explore the role of adenosine monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor α (PPARα） signal pathway in the regulation of SCAD during the development of cardiac hypertrophy. METHODS：The optimal sequence of SCAD interference was chosen by Western blotting and real-time PCR. The cardiomyocytes were treated with fenofibrate (10 μmol/L) for 24 h and subsequently stimulated with the optimal sequence of SCAD interference. The changes of SCAD expression at mRNA and protein levels, the enzyme activity of SCAD, the cardiomyocyte surface area and free fatty acids were determined. Using real-time PCR for analyzing the markers of cardiac hypertrophy, the mRNA expression of atrial natriuretic factor (ANF） and brain natriuretic peptide (BNP) was detected to judge the development of cardiac hypertrophy. The cardiomyocytes were treated with fenofibrate (10 μmol/L) or AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, 0.5 mmol/L) for 30 min and subsequently stimulated with phenylephrine (PE, 20 μmol/L) for 24 h. The changes of cardiomyocyte surface area, free fatty acids, and the expression of SCAD, PPARα and p-AMPKα (T172) at mRNA and protein levels were observed. RESULTS：The effect of optimal sequence siRNA-1186 and PE on the cardiomyocytes was the same. Compared with control group, the expression of ANF and BNP at mRNA level, the cardiomyocyte surface area and free fatty acids were increased obviously in siRNA-1186 group. After pretreated with fenofibrate (10 μmol/L), the expression of PPARα and SCAD, and the enzyme activity of SCAD were significantly increased, while the free fatty acids were decreased, indicating that fenofibrate prevented the development of cardiac hypertrophy induced by knockdown of SCAD. Compared with control group, the expression of SCAD, PPARα and p-AMPKα (T172) at mRNA and protein levels was significantly down-regulated, and the enzyme activity of SCAD was obviously decreased in PE group. Compared with PE group, the expression of SCAD, PPARα and p-AMPKα (T172) was significantly up-regulated, and the cardiomyocyte surface area and the content of free fatty acids were obviously decreased in the cardiomyocytes pretreated with fenofibrate or AICAR for 30 min. CONCLUSION：Down-regulation of SCAD is related to the cardiac hypertrophy and energy metabolism. AMPK/PPARα/SCAD signaling pathway may regulate cardiac hypertrophy directly.     

Role of calreticulin-induced mitochondrial damage in high glucose-induced apoptosis of myocardial cells

AIM: To observe the effect of high glucose on the protein expression of calreticulin (CRT) and its association with cell apoptosis and mitochondrial dysfunction in the cardiomyocytes. METHODS: AC-16 cardiomyocytes were randomly divided into normal glucose group, high glucose group, high glucose+ CRT siRNA group and isotonic control group. The cell apoptotic rate, reactive oxygen species (ROS), mitochondrial membrane potential level, respiratory enzyme activity, and protein expression of CRT were observed. RESULTS: Compared with the cardiomyocytes in normal glucose group, the apoptotic rate and ROS production of cardiomyocytes increased in high glucose group, accompanying with the decreases in the mitochondrial membrane potential level and enzyme activitiy of the respiratory chain. The protein expression of CRT was significantly increased in high glucose group. However, compared with high glucose group, high glucose+ CRT siRNA decreased the expression of CRT and attenuated the damage of mitochondria, but CRT siRNA did not reduce the ROS level in cardiomyocytes. CONCLUSION: High glucose brings about CRT over-expression to induce mitochondrial injury, thus increasing myocardial apoptosis.     

Effects of PPARα activation on AngⅡ-induced cardiomyocyte hypertrophy and interaction of NFATc4 with p65-NFκB

AIM：To investigate the effects of fenofibrate on angiotensin Ⅱ (AngⅡ)-induced cardiomyocyte hypertrophy. METHODS：Primary neonatal cardiomyocytes were pretreated with fenofibrate (10 μmol/L) for 1 h followed by stimulation with AngⅡ (100 nmol/L). The mRNA levels of ANF, BNP and β-MHC were measured by real-time PCR. Western blotting was employed to determine the nuclear translocations of NFATc4 and p65-NFκB. Co-immunoprecipitation was used to investigate the interaction of NFATc4 with p65-NFκB in the nucleus of cardiomyocytes. In addition, the DNA binding activity of NFATc4 on the BNP promoter was determined by EMSA. RESULTS：Fenofibrate significantly inhibited AngⅡ-induced cardiomyocyte hypertrophy. Fenofibrate treatment inhibited the nuclear translocations of NFATc4 and p65-NFκB, as well as the interactions of NFATc4 with p65-NFκB in the nucleus of cardiomyocytes induced by AngⅡ. Fenofibrate inhibited the binding activity of NFATc4 with the BNP promoter, which was strengthened by AngⅡ. CONCLUSION： Fenofibrate enhances the interaction of NFATc4 with PPARα, decreases the interaction of NFATc4 with p65-NFκB in the nucleus of cardiomyocytes, and inhibits the DNA binding activity of NFATc4 induced by AngⅡ, which may be the important mechanisms of fenofibrate on inhibiting cardiac hypertrophy.     

Role of PARP-2 in cardiac hypertrophy in rats

AIM: To investigate the expression of poly(ADP-ribose) polymerase-2 (PARP-2) during rat cardiac hypertrophy in vitro and in vivo, and to explore the effects of PARP-2 on the cardiac hypertrophy. METHODS: Abdominal aortic coarctation (AAC) was performed to establish a model of pressure overload-induced cardiac hypertrophy in SD rats. The expression of PARP-2 at mRNA and protein levels in the myocardium was determined by real-time PCR and Western blot. The hypertrophy model of the cardiomyocytes was induced by treating the cells with angiotensin Ⅱ (AngⅡ). PARP-2 was knocked down by siRNAs in neonatal rat cardiomyocytes and the cardiomyocyte hypertrophy was evaluated by measuring the mRNA levels of ANF, BNP, and β-MHC and the cellular surface area. RESULTS: The expression of PARP-2 at mRNA and protein levels was both increased in the AAC rats as compared with those in the sham animals. The expression of PARP-2 at mRNA and protein levels was also increased in a time- and concentration-dependent manner in AngⅡ-induced hypertrophy model of the cardiomyocytes. In the neonatal rat cardiomyocytes, knockdown of PARP-2 expression by siRNA attenuated AngⅡ-induced cardiac hypertrophy of the cardiomyocytes, indicating that endogenous PARP-2 played a positive regulatory role in cardiac hypertrophy. CONCLUSION: The mRNA and protein levels of PARP-2 increase in the in vitro and in vivo models of cardiac hypertrophy. Knockdown of PARP-2 protects cardiomyocytes from hypertrophy.     

Changes of cardiac function,RAGE expression and calcium dysregulation in type 2 diabetic rats

AIM: To investigate the changes of cardiac structure and function in rats with type 2 diabetic mellitus (T2DM), and to explore the mechanisms underlying diabetic cardiomyopathy. METHODS: The cardiac structure and function were measured by echocardiography in Zucker diabetic fatty (ZDF) rats and their control Zucker lean (ZL) rats. The size of the cardiomyocytes was determined by wheat germ agglutinin staining. The protein expression of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), receptor for advanced glycation end products (RAGE), L-type cal-cium channel α1C subunit (Ca_V1.2) and Orai1 was assessed by Western blot. RESULTS: Compared with the ZL control rats, the thickness of left ventricular wall, ejection fraction (EF), fractional shortening (FS) and the sizes of cardiomyocytes were significantly increased, and diastolic function was decreased in the ZDF rats (P<0.05). The protein expression of β-MHC, ANP, RAGE and Orai1 was increased, while the expression of Ca_V1.2 was decreased in ZDF rats (P<0.05). CONCLUSION: T2DM rats show the prominent features including cardiomyocyte hypertrophy, ventricular hypertrophy and compensatory enhancement of cardiac function, and the Ca²⁺ handling and increase in RAGE expression may play important roles in the processes.     

Effects of P38 MAPK signaling pathway on anacardic acid attenuating mouse cardiomyocyte hypertrophy induced by phenylephrine

AIM: To investigate the roles of P38 mitogen-activated protein kinase (P38 MAPK) in the process of anacardic acid (AA) attenuating mouse cardiomyocyte hypertrophy induced by phenylephrine (PE). METHODS: Cardiomyocyte hypertrophy was induced by PE in primary neonatal mouse myocardial cells. According to random number table method, the experiments were designed in 6 groups as following: control group, PE+ DMSO group, PE group, PE+ AA group, PE+ AA+ P38 inhibitor group and PE+ P38 inhibitor group. Mouse myocardial cells were collected after intervention for 48 h. The protein levels of p-P38, acetylated histone H3 at lysine 9 (H3K9ac) and atrial natriuretic peptide (ANP) were determined by Western blot. The interaction between p-P38 and H3K9ac was verified by co-immunoprecipitation (Co-IP). The mRNA expression of myocyte enhancer factor 2C (MEF2C) were tested by RT-qPCR. Mouse myocardial cell surface area was observed by immunofluorescence staining. RESULTS: The results of Western blot showed that the protein levels of p-P38 and H3K9ac in PE group were significantly increased compared with control group (P<0.05), and the levels of MEF2C mRNA and ANP protein in PE group were apparently increased compared with control group (P<0.05). However, P38 inhibitor and histone acetylase inhibitor AA attenuated P38 phosphorylation and H3K9 acetylation induced by PE, and down-regulated the over-expression of MEF2C and ANP in the mouse myocardial cells (P<0.05). The results of immunofluorescence staining showed that PE apparently increased mouse myocardial cell surface area (P<0.05), while P38 inhibitor and AA attenuated cardiomyocyte hypertrophy induced by PE (P<0.05). CONCLUSION: The mechanism of AA attenuating cardiomyocyte hypertrophy induced by PE may be related to the regulation of histone acetylation modification imbalance mediated by P38 MAPK signaling pathway.     

Change of subunit of NADPH oxidation enzyme complex nox-1 protein in cardiocyte hypoxia-reoxygenation injury and the role of cardiotrophin-1

AIM: To observe the change of subunit of NADPH oxidation enzyme complex nox-1 protein in cardiocyte hypoxia-reoxygenation injury and the role of cardiotrophin-1.METHODS: Cardiomyocytes from the hearts of 1-3 d old neonatal rats were prepared by a modified method. Five groups were included in the study: control; hypoxia/reoxygenation; hypoxia/reoxygenation+CT-1; CT-1+hypoxia/reoxygenation+LY294002 (PIK3/Akt inhibitor); CT-1+hypoxia/reoxygenation+PD98059 (ERK inhibitor); CT-1+hypoxia/reoxygenation+DMSO. The concentration of CT-1 was 10 μg/L. The survival rate of myocytes was evaluated by MTS method. Apoptosis, mitochondrial permeability transition pore (Δψm) and reactive oxygen species (ROS) were detected by flow cytometry. Nox-1 protein was determined by Western blotting.RESULTS: Apoptosis of cardiomyocytes and the level of ROS (19.7%±1.4% vs 2.1%±0.5%, 14.07%±1.25% vs 3.54%±0.86%, P<0.05) increased markedly after hypoxia/reoxygenation, but cardiomyocyte survival rate and the level of Δψm (40.55%±4.25% vs 86.28%±7.15%, P<0.01) decreased significantly. The expression of nox-1 protein was upregulated markedly. With CT-1 intervention, cardiomyocyte survival rate increased markedly, apoptosis, both ROS and expression of nox-1 protein reduced significantly. The level of Δψm increased obviously. The effect of CT-1 was inhibited by LY294002. No significant effect was observed on cells survival in DMSO group, which confirmed that LY294002 was specifically involved in blocking the protective effect of CT-1.CONCLUSION: The expression of subunit of NADPH oxidation enzyme complex nox-1 protein is upregulated markedly in cardiocyte hypoxia-reoxygenation injury. CT-1 protects cardiac cells against hypoxia-reoxygenation injury by downregulating the expression of nox-1 protein to decrease the level of ROS.     

Vitamin D prevents high glucose-induced human umbilical vein endothe-lial cell apoptosis by inhibition of prolyl isomerase 1-mediated mitochon-drial oxidative stress

AIM: To observe the effects of vitamin D on the apoptosis, prolyl isomerase 1 (Pin1) protein expression and activity, mitochondrial translocation of p66Shc, and reactive oxygen species (ROS) production in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and to explore the role of vitamin D receptor (VDR) in these processes. METHODS: HUVECs were treated with high glucose (33 mmol/L) in the presence or absence of vitamin D or Pin1 inhibitor juglone. The cell apoptosis was measured by flow cytometry and TUNEL staining. Intracellular ROS levels were examined by flow cytometry and fluorescence microscopy. The protein levels of Pin1, p66Shc, p-p66Shc, mitochondria to cytoplasm ratio of p66Shc, and caspase-3 in HUVECs were measured by Western blot. Pin1 activity in HUVECs lysate was assessed by a commercial kit. Knockdown of VDR by siRNA was conducted to evaluate the role of VDR in the regulatory effects of vitamin D on Pin1 protein expression and activity in HUVECs under high-glucose condition. RESULTS: Vitamin D suppressed the apoptosis and intracellular ROS generation of HUVECs induced by high glucose (P<0.05). Vitamin D inhibited high glucose-induced upregulation of Pin1 protein expression and activity (P<0.05). Vitamin D inhibited the phosphorylation and mitochondrial translocation of p66Shc and caspase-3 protein expression induced by high glucose (P<0.05). Knockdown of VDR by siRNA abolished the inhibitory effects of vitamin D on high glucose-induced upregulation of Pin1 protein expression and activity. CONCLUSION: Vitamin D alleviates high glucose-induced endothelial cell apoptosis by inhibition of Pin1 protein expression and activity, and attenuation of p66Shc-mediated mitochondrial oxidative stress, which are dependent on VDR activation.     

Effects of PPARβ and NO on high glucose and insulin-induced cardiomyocyte hypertrophy

AIM: To investigate the role of peroxisome proliferator-activated receptor β(PPARβ)-nitric oxide(NO) signal pathway in cardiomyocyte hypertrophy induced by high glucose(25.5 mmol/L) and insulin(0.1 μmol/L)(HGI). METHODS: The cardiomyocyte hypertrophy was characterized in rat primary cardiomyocytes by measuring the cell surface area, protein content, and the mRNA expression of atrial natriuretic factor(ANF). The mRNA and protein expression were measured by real-time PCR and Western blotting, respectively. The activity of NO synthase(NOS) and NO content were measured by a reagent kit through ultraviolet spectroscopy. RESULTS: HGI induced profound change of hypertrophic morphology, and significantly increased the cell surface area, protein content and mRNA expression of ANF(P<0.01), but decreased the expression of PPARβ at mRNA and protein levels(P<0.05). At the same time, the expression of inducible NOS(iNOS) was obviously elevated(P<0.01), which occurred in parallel with the rising NOS activity and NO concentration(P<0.01). GW0742(1 μmol/L), a selective PPARβ agonist, inhibited the cardiomyocyte hypertrophy induced by HGI(P<0.01), and up-regulated the expression of PPARβ at both mRNA and protein levels. Meanwhile, GW0742 also inhibited the increases in iNOS expression, NOS activity, and NO content induced by HGI, which were abolished by GSK0660(1 μmol/L), a selective PPARβ antagonist(P<0.01). CONCLUSION: PPARβ down-regulation and the following iNOS-NO activation are involved in the cardiomyocyte hypertrophy induced by HGI.     

Involvement of Sirt1 in mouse myocardial hypertrophy induced by isorenin

AIM: To investigate the expression and function of sirtuin 1 (Sirt1), one of the class III histone deacetylases, in hypertrophied mouse myocardium induced by isorenin (ISO). METHODS: The Kunming mice were randomly divided into 3 groups: control group, ISO group and ISO+nicotinamide (NAM) group. The myocardial hypertrophy was induced by dorsal subcutaneous injection of isorenin. Nicotinamide, an inhibitor against Sirt1, was given by peritoneal injection. Heart weight index (heart weight/body weight), hematoxylin and eosin staining, transmission electron microscope and mRNA expression of brain natriuretic peptide (BNP) were observed to identify the myocardial hypertrophy. The expression of Sirt1 in mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. RESULTS: Compared to control group, the results of heart weight index, hematoxylin and eosin staining, the observation of transmission electron microscopy and mRNA expression of BNP showed that the mouse myocardial hypertrophy was induced by isorenin successfully. The Sirt1 expression was increased in hypertrophy model group (P<0.01 vs control group). Treatment with nicotinamide inhibited the cardiac hypertrophy induced by ISO (P<0.05 vs ISO group) and decreased the expression of Sirt1 (P<0.01 vs ISO group). CONCLUSION: Activation of Sirt1 might be involved in the process of myocardial hypertrophy stimulated by isorenin in mice.     

Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human umbilical vein endothelial cells

AIM:To explore the effect of atorvastatin on high glucose-induced oxidative stress and underlying mechanisms in human endothelial cells. METHODS:Human umbilical vein endothelial cells(HUVECs) were cultured in medium 199 containing normal concentration of glucose(5.5 mmol/L). For high glucose treatment, glucose solution was added to the final concentration of 25 mmol/L. Reactive oxygen species(ROS) were detected by flow cytometry and confocal microscopy. The activity of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase was measured by lucigenin assay. Phosphorylated protein kinase C(PKC) and the expression levels of NADPH oxidase subunits Nox4 and Nox2/gp91^phox were determined by quantitative real-time PCR and immunoblotting. RESULTS:High glucose increased ROS production, NADPH oxidase activity and the expression of Nox4 and Nox2/gp91^phox subunits. Treatment of endothelial cells with atorvastatin resulted in significant inhibition(in a concentration-dependent manner) of high glucose-induced ROS production, NADPH oxidase activation and the expression of Nox4 and Nox2/gp91^phox subunits. PKC inhibitor showed a similar effect to that of atorvastatin on high glucose-induced oxidative stress. Furthermore, atorvastatin rapidly inhibited high glucose-induced activation of protein kinase C, an upstream activator of NADPH oxidase. CONCLUSION:PKC is involved in high glucose-induced oxidative stress in HUVECs. Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human endothelial cells.     

Cyclosporine A suppresses the hypertrophic effect of neuropeptide Y in rat cardiomyocytes

AIM:To investigate the effect of cyclosporine A on rat cardiomyocyte hypertrophy caused by neuropeptide Y (NPY).METHODS:Cardiomyocytes of neonatal Wistar rats were cultured with NPY or NPY together with CsA. For assessing protein synthesis rate and c-jun mRNA expression in cardiomyocytes, the methods of [³H]-Leu incorporation and RT-PCR were used.RESULTS:(1) [³H]-Leu incorporation in cardiomyocytes: [³H]-Leu incorporation in NPY (10 nmol/L) group was higher than that in control group, but there were no distinct changes between two groups. To compare with control group, [³H]-Leu incorporation in NPY (100 nmol/L) group were increased significantly (P＜0.05). There was no significant change between control group and CsA group; (2) c-jun mRNA expression in cardiomyocytes: RT-PCR production of c-jun mRNA in NPY group was enhanced considerably compared with CsA group and control group (P＜0.01). There was no significant change between CsA group and control group.CONCLUSIONS:NPY can induce cardiomyocyte hypertrophy. Cyclosporine A (inhibitor of CaN) can blunt the effect of NPY, suggesting that the Ca²⁺/CaM-dependent calcineurin (CaN) signaling pathway plays an important role in it.