NRF2 attenuates oxidative stress and lysosomal dysfunction in doxorubicin-induced H9C2 cells

AIM:To study the effect of nuclear factor E2-related factor 2 (NRF2) on oxidative stress injury and lysosomal dysfunction in doxorubicin (DOX)-induced rat myocardial H9C2 cells. METHODS:The H9C2 cells were treated with DOX. The expression of NRF2 at mRNA and protein levels was determined by real-time PCR and Western blot. The H9C2 cells stably over-expressing NRF2 were established by lentiviral infection. Real-time PCR and Western blot were used to identify the efficiency of over-expression. After DOX treatment, the cell viability was measured by CCK-8 assay, the activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the content of malondialdehyde (MDA) in the cell supernatant were detected. FITC-dextran was used to analyze lysosomal pH, and the protein expression of lysosomal-associated membrane protein 1 (LAMP1) and cathepsin B was determined by Western blot.RESULTS:The expression of NRF2 at mRNA and protein levels in DOX-treated H9C2 cells was significantly decreased (P<0.05). Over-expression of NRF2 significantly up-regulated the mRNA and protein expression of NRF2 in DOX-treated H9C2 cells (P<0.05). After DOX treatment, the cell viability was decreased, and LDH activity was increased. The activity of SOD, GSH-Px and CAT was decreased, and the content of MDA was increased (P<0.05). The lysosomal pH was increased, and the protein expression of LAMP1 and cathepsin B decreased (P<0.05). Over-expression of NRF2 increased the cell viability, decreased LDH activity, increased the activity of SOD, GSH-Px and CAT, and decreased the content of MDA in cell supernatant (P<0.05). Over-expression of NRF2 also decreased the lysosomal pH, and increased the protein expression of LAMP1 and cathepsin B (P<0.05). CONCLUSION:DOX inhibits the expression of NRF2 in the myocardial H9C2 cells. Over-expression of NRF2 attenuates oxidative stress and lysosomal dysfunction in the H9C2 cells induced by DOX.     

ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway， thus promoting the apoptosis of cardiomyocytes.     

Effect of ROCK1 and ROCK2 silencing by shRNA on hypoxia-induced apoptosis of rat cardiomyocytes

AIM: To investigate the effects of Rho-associated coiled-coil protein kinase-1 (ROCK1) and ROCK2 on apoptosis induced by hypoxia in rat cardiomyocytes. METHODS: Rat cardiomyocytes were cultured primarily and identified using an antibody targeting α-actin of striated muscle. ROCK1-shRNA and ROCK2-shRNA were transiently transfected into the cells by liposome. After 48 h, these cells were subject to hypoxia for 6 h. The cells were divided into 5 groups: blank control group, hypoxia group, hypoxia+negative control shRNA group, hypoxia+ROCK1-shRNA group and hypoxia+ROCK2-shRNA group. The beating frequency and rhythm of the cardiomyocytes were assessed by microscopy. The activity of lactate dehydrogenase (LDH) in the cell culture supernatants was detected by automatic biochemical analyzer. The cell survival rate was analyzed by the method of MTT. The cell apoptotic rate was assessed by flow cytometry. Western blotting was used to determine the expression of ROCK1, ROCK2, caspase-3 and p-PI3K. RESULTS: The primary culture of the cardiomyocytes was successful. Western blotting results showed that the transfection of ROCK1-shRNA or ROCK2-shRNA decreased the expression of ROCK1 or ROCK2 in the cardiomyocytes. Hypoxia slowed down the beat frequency of the cardiomyocytes, also made the rhythm disorder. Hypoxia increased the release of LDH and decreased the cell survival rate. Flow cytometry results showed that hypoxia increased the cell apoptotic rate. Hypoxia increased the expression of caspase-3 and decreased the expression of p-PI3K. Transfection of ROCK1-shRNA and ROCK2-shRNA into the cardiomyocytes reduced all the effects of hypoxia mentioned above. CONCLUSION: Down-regulation of ROCK1 and ROCK2 expression suppresses the apoptosis of rat cardiomyocytes induced by hypoxia. The mechanism is associated with the inhibition of caspase-3 activation and the up-regulation of p-PI3K expression.     

Role of voltage-dependent anion channel in cardiomyocytes subjected to anoxia/reoxygenation

AIM: To investigate the role of voltage-dependent anion channel (VDAC) in H9c2 cardiomyocytes subjected to anoxia/reoxygenation (A/R).METHODS: The shRNAs targeting VDAC1 mRNA were inserted into pSUPER plasmid.H9c2 cells were randomly divided into 5 groups as follows: control group, A/R group, anoxia preconditioning (APC) group, pSUPER-VDAC1-A/R group and pSUPER-A/R group. The expression of VDAC1 was detected by Western blotting. Cellular injury was evaluated by measuring the cell viability and the release of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK). The mitochondria membrane potential was determined by flow cytometry.RESULTS: VDAC1 expression was up-regulated in A/R group and was inhibited in APC group. Similarly, down-regulation of VDAC1 expression by shRNA protected H9c2 cells from A/R injury. Moreover, we found that, with silencing VDAC1 expression, mitochondrial membrane potential was well preserved in H9c2 cells subjected to A/R.CONCLUSION: Down-regulation of VDAC1 protects H9c2 cells against A/R injury and its possible mechanism appears to be related to the regulation of mitochondial permeability transition pore opening.     

Effect of breviscapin on Ito and IK1 channel current in isolated ventricular myocytes of rats

AIM: The effects of breviscapin on transient outward potassium current (I_to) and inward rectifier potassium current (I_K1) channel in isolated ventricular myocytes of rats were determined. The mechanism of breviscapin at the ionic channel level was explored. METHODS: Single ventricular myocyte of rats was isolated by enzymatic dissociation. The whole-cell patch-clamp recording technique was used to record the change of I_to and I_K1 channel current influenced by breviscapin. RESULTS: Breviscapin decreased the I_to channel current in a dose-dependent and voltage-dependent manner in ventricular myocytes of rats. (1) The current-voltage curve was significantly decreased. Breviscapin at concentrations of 0.02, 0.05, 0.08, 0.10 g·L^-1 respectively decreased I_to current density (10.07±0.50)%, (27.47±2.36)%, (42.72%±2.50)% and (56.09±5.60)%. I_to was inhibited in a voltage-dependent manner, showing a significant attenuating effect at test potentials from 0 to + 50 mV. (2) The I_to activation curve, the activation curve and the recovery curve were not altered. (3) Breviscapin did not affect I_K1. CONCLUSION: Breviscapin concentration-dependently decreases I_to channel current in ventricular myocytes of rat.     

Adeno-associated virus type 9-mediated p65 silencing inhibits angiotensin II-induced apoptosis of H9c2 cells

AIM: To study the effect of p65 gene silencing by adeno-associated virus type 9 (AAV9)-mediated RNA interference on angiotensin Ⅱ (Ang Ⅱ; 10^-6 mol/L for 24 h)-induced apoptosis of rat ventricular H9c2 myocytes, and to elucidate the possible mechanism. METHODS: The H9c2 cells were transfected with rAAV9-eGFP and rAAV9-eGFP-NF-κB p65-siRNA at multiplicity of infection (MOI)=4×10⁶ vg/cell. eGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of eGFP positive cells was determined by flow cytometry. The expression of p65 was determined by Western blot. CCK-8 assay was used to measured the viability of transfected H9c2 cells. The apoptosis of the cells transfected with the virus and with Ang Ⅱ stimulation was analyzed by flow cytometry. RESULTS: The cells began to exhibit eGFP expression on the 2nd day after transfection. The fluorescence intensity was increased over the time of transfection. eGFP expression reached the maximum on the 5th day, and the transfection efficiency was (52.7±1.9)% at this time point. Compared with blank control group, no significant effect of AAV9 on the viability of H9c2 cells was observed. In resting state, p65 in the H9c2 cells had a certain activity. After Ang Ⅱ stimulation, the activity of p65 was obviously increased, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited the expression of p65. The apoptosis of H9c2 cells in Ang Ⅱ stimulation group was significantly higher than that in blank control group, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited apoptosis of H9c2 cells. CONCLUSION: Transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibits the expression of p65 gene of NF-κB pathway in the H9c2 cells without causing cell growth inhibition, and reduces the apoptosis induced by Ang Ⅱ.     

PERK signal pathway is involved in hypoxia-induced endoplasmic reti-culum stress and apoptosis in cultured cardiac myocytes

AIM:To investigate the role of endoplasmic reticulum(ER) stress in the process of hypoxia-induced neonatal rat myocardial injury through PERK signal pathway. METHODS:Neonatal rat cardiac myocytes were randomly divided into control group and hypoxia 1 h, 4 h, 8 h, 12 h and 24 h groups. Cell viability was evaluated by determining the intracellular content of ATP. Apoptosis was measured by high-content analysis(HCA) cell imaging system. The protein levels of GRP78, calreticulin, p-PERK, p-eIF2α, ATF4 and CHOP were detected by Western blotting at different time points. The primary cultured neonatal rat cardiac myocytes were treated with an agonist of PERK pathway salubrinal and the cell apoptosis was observed under hypoxia. RESULTS:In the early phase, hypoxia induced an increase in the expression of calreticulin and GPR78. In the middle phase of hypoxia, the levels of p-PERK, p-eIF2α and ATF4 were increased. In the later phase of hypoxia, increased CHOP level was also observed. Salubrinal effectively protected the cardiac myocytes from hypoxic injury. CONCLUSION:Hypoxia activates ER stress in cardiac myocytes and also activates PERK signal pathway. PERK signaling protects cardiac myocytes from hypoxic damage in the early stage and triggers apoptosis of the cells in the later phase.     

Effect of advanced glycation end products on inflammation in cultured cardiomyocytes

AIM: To investigate the effect of advanced glycation end products on inflammation in cultured cardiomyocytes.METHODS: Primary cardiomyocytes were isolated from Sprague-Dawley neonatal (1 to 2 days old) rats ventricles.The insulin resistant cardiomyocyte model was established.Neonatal rat ventricular myocytes were exposed to AGEs for 24 hours.TNF-α mRNA and PPAR-γ mRNA expressions were determined by RT-PCR.Activation of NF-κB in the cells was examined by using immunocytochemistry.The ultrastructure of the cells was detected by transmission electron microscope.RESULTS: The exprssion of TNF-α mRNA and the activation of NF-κB increased,the expression of PPAR-γ mRNA decreased compared with control group (P<0.05).The differences among different AGE-BSA groups were significant (P<0.05).The numbers of chondriosome and smooth endoplasmic reticulum increased.CONCLUSION: AGEs significantly increase TNF-α mRNA expression and NF-κB activation,and restrain the expression of PPAR-γ mRNA.These data suggest that AGEs play an important role in the onset of diabetic cardiomyopathy.