Expression and effect of miR-139-3p in apoptotic neonatal rat cardiomyocytes induced by hypoxia

AIM:To investigate the expression and clinical significance of microRNA-139-3p (miR-139-3p) in the apoptosis model of cardiomyocytes induced by hypoxia. METHODS:Under normal and hypoxic conditions, the expression of miR-139-3p in neonatal rat cardiomyocytes was detected by RT-qPCR. miR-139-3p inhibitor and miR-139-3p inhibitor negative control were transfected into the primary neonatal rat cardiomyocytes. The transfected cardiomyocytes were cultured in closed anoxic box (95% N₂ and 5% CO₂) at 37℃ for 12 h. Flow cytometry and Western blot were used to determine the apoptosis of cardiomyocytes. RESULTS:After hypoxia for 12 h, the expression level of miR-139-3p and the apoptotic rate of the cardiomyocytes in hypoxia group were significantly higher than those in normal group (P<0.05). Moreover, compared with the miR-139-3p inhibitor negative control group, the apoptotic rate of the cardiomyocytes was significantly decreased in miR-139-3p inhibitor group (P<0.05). CONCLUSION:The expression of miR-139-3p is signi-ficantly increased in apoptotic neonatal rat cardiomyocytes induced by hypoxia. Inhibition of miR-139-3p expression reduces hypoxia-induced apoptosis of cardiomyocytes.     

miR-323 regulates hypoxia-induced apoptosis of H9C2 cardiomyocytes through FGF9

AIM: To investigate the effect of microRNA-323 (miR-323) on the apoptosis of hypoxia-induced rat H9C2 cardiomyocytes and its mechanism. METHODS: The hypoxic injury model was established in the H9C2 cells. Anti-miR-323, pcDNA-FGF9 and si-FGF9 were transfected into the H9C2 cells and cultured under hypoxic condition for 48 h. The expression of miR-323 was detected by qPCR. The protein levels of fibroblast growth factor 9 (FGF9), cleaved caspase-3, c-Jun N-terminal kinase (JNK) and p-JNK were determined by Western blot. The cell viability was measured by MTT assay, and the apoptosis was analyzed by flow cytometry. The method of bioinformatics was applied to predict the target gene of miR-323, and dual-luciferase reporter assay was used for further validation. RESULTS: Hypoxia greatly reduced the viability of H9C2 cells at 12 h, 24 h and 48 h (P<0.05), and remarkably increased apoptotic rate and the protein level of cleaved caspase-3 in a time-dependent manner (P<0.05). The expression of miR-323 and the protein level of p-JNK were up-regulated and the expression of FGF9 was down-regulated in the H9C2 cells exposed to hypoxia (P<0.05). Down-regulation of miR-323 and over-expression of FGF9 obviously increased the viability of the H9C2 cells exposed to hypoxia, and decreased the apoptotic rate and the protein level of cleaved caspase-3 (P<0.05). FGF9 was the target gene of miR-323. Down-regulation of FGF9 reversed the attenuating effect of down-regulation of miR-323 on hypoxia-induced H9C2 cell injury. miR-323 regulated FGF9 and affected p-JNK level. CONCLUSION: miR-323 affects the viability and apoptosis of H9C2 cardiomyocytes under hypoxia by targeting FGF9 and regulating JNK signaling pathway.     

Effect of Shenshuguanxin granula on coronary circulation in rats with myocardial infarction

AIM：To explore the effect of traditional Chinese medicine Shenshuguanxin granula on coronary circulation in a rat model of myocardial infarction (MI). METHODS：SD rats (n=50, SPF grade) were randomly divided into 5 groups (n=10):sham group, MI group, and high-dose, middle-dose and low-dose Shenshuguanxin granula treatment groups. The rat MI model was established by ligation of the coronary artery. The cardiac markers, small and medium-sized blood vessels ［microvessel count (MVC) value］ in the infarct zone, and platelet endothelial cell adhesion mo-lecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) expression in the infarct border zone were measured. RESULTS：After 4 weeks of coronary artery ligation, the significant increases in MVC in the infarct zone, and the expression of PECAM-1 and VEGF in the infarct border zone were detected compared with sham group (P<0.05). The differences of cardiac markers between MI group and other groups were insignificant (P>0.05). CONCLUSION:Shenshuguanxin granula improves coronary circulation in the rats with myocardial infarction by increasing the expression of PECAM-1 and VEGF, and promoting small and medium-sized angiogenesis.     

Proctective effect of microRNA-30a on regulating beclin-1 expression in hypoxia-reoxygenated neonatal rat cardiomyocytes

AIM: To explore the potential mechanism of microRNA-30a (miR-30a) overexpression in neonatal rat cardiomyocytes during hypoxia/reoxygenation (H/R). METHODS: The miR-30a overexpression was induced in primary neonatal rat cardiomyocytes by lentivirus transfection. The cardiomyocytes were divided into 5 groups: normal group, H/R group, LV-GFP+H/R group, LV-GFP-miR-30a+H/R group and 3-methyladenine(3-MA)+H/R group. The expression level of miR-30a after lentivirus transfection and H/R was determined by real-time PCR, while the protein levels of LC3 and Beclin-1 after H/R and lentivirus transfection were detected by Western blotting. The cardiomyocyte death after H/R were measured by TUNEL and PI staining. RESULTS: Compared with LV-GFP group, significant down-regulation of Beclin-1 protein level was observed in cardiomyocytes with miR-30a overexpression, while the protein levels of Beclin-1 and LC3 in the cardiomyocytes with miR-30a overexpression were down-regulated after H/R, and apoptosis of these cells were significantly decreased after H/R. CONCLUSION: The protein level of Beclin-1 is down-regulated in cardiomyocytes with miR-30a overexpression. Inhibition of autophagy decreases the cardiomyocyte death after H/R.     

Regulatory effect of miR-214-5p on myocardial injury and immune response in ischemia-reperfusion rats by targeting PAK4

AIM: To investigate the effect of microRNA-214-5p (miR-214-5p) on myocardial injury and immune response in rats with ischemia-reperfusion (I/R) by targeting p21-activated protein kinase 4 (PAK4). METHODS: The rats were divided into sham group, I/R group, Ad-Scramble group, and Ad-miR-214 group (n=9). Adenovirus was injected into 6 different sites on the anterior wall of the left ventricle of the rats. Four days later, the I/R model was constructed by suturing the left anterior descending coronary artery. The expression level of miR-214 was detected by RT-qPCR. Myocardial injury was observed by HE staining. The levels of heart damage markers (CK-MB, Mb, and cTnI) and inflammatory factors (IL-6, IL-1β and TNF-α) were measured by ELISA. The rate of cardiomyocyte apoptosis was analyzed by flow cytometry. The content of MDA and the activity of SOD were detected by commercially available kits. Target genes were predicted by genetic software and verified by dual-luciferase reporter assay. The protein levels of caspase-3, caspase-9, Bcl-2, Bax, PAK4, p-Akt and p-mTOR were determined by Western blot. RESULTS: miR-214 was down-regulated in the cardiomyocytes of I/R rats (P<0.01). Over-expression of miR-214-5p attenuated myocardial injury in the I/R rats, down-regulated the expression of CK-MB, Mb and cTnI, decreased the apoptotic rate of cardiomyocytes, up-regulated the expression of Bcl-2, down-regulated Bax, caspase-3 and caspase-9 expression, increased SOD activity, and decreased the content of MDA, IL-6, IL-1β and TNF-α (P<0.01). The binding sites of miR-214-5p and PAK4 were pre-sent in the 3’-UTR, and over-expression of miR-214-5p up-regulated the protein levels of PAK4, p-Akt and p-mTOR (P<0.01). CONCLUSION: miR-214-5p over-expression attenuates myocardial injury in I/R rats by targeting PAK4, inhibits cardiomyocyte apoptosis, oxidative stress and inflammation, and activates the PI3K/Akt/mTOR pathway.     

Relationship of microRNA-133a and TGF-β1 protein in myocardial tissues of spontaneously hypertensive rats

AIM：To observe the changes of microRNA-133a and transforming growth factor β1 (TGF-β1) protein in the myocardium of spontaneously hypertensive rats (SHR). METHODS：Male SHR (18 weeks old, n=12) and male Wistar-Kyoto rats (WKY, 18 weeks old, n=12) served as SHR group and control group, respectively. Caudal arterial blood pressure was detected by a noninvasive blood pressure measurement and analysis system. Myocardial collagen volume fraction (CVF) and perivascular collagen area ratio (PVCA) were determined by Masson staining. The level of miR-133a in the heart was detected by real-time quantitative PCR. The protein level of TGF-β1 in the heart was also analyzed by the methods of immunohistochemisty and Western blotting. RESULTS：Compared with control group, systolic and diastolic blood pressure, CVF and PVCA significantly increased, the expression of TGF-β1 protein was significantly up-regulated, and the level of miR-133a was significantly reduced in SHR group. In SHR group, the expression of miR-133a was decreased to (23.9±4.6)% in control group. A negative correlation between the levels of miR-133a and TGF-β1 protein in SHR group was observed (r=-0.791, P<0.01). CONCLUSION：The level of miR-133a is down-regulated along with the up-regulation of TGF-β1 protein expression and collagen synthesis in the myocardial tissues of SHR. miR-133a and TGF-β1 may be involved in myocardial fibrosis in SHR.     

Panax quinquefolium saponins attenuate hypoxia/reoxygenation-induced injury in rat cardiomyocytes

AIM： To investigate the effects of Panax quinquefolium saponins (PQS) and calcineurin (CaN) signal pathway on cardiomyocyte injury induced by myocardial hypoxia/reoxygenation(H/R). METHODS：Cultured cardiomyocytes isolated from neonatal Sprague-Dawley rats were used to establish the H/R model. The cells were transfected with pCDB-CaN plasmid to overexpress CaN, or exposed to the CaN inhibitor FK506 to interfere the CaN expression. The cardiomyocytes were divided into control group, H/R group, PQS+H/R group, CaN+PQS+H/R group, pCDB+PQS+H/R group and FK506+PQS+H/R group. The apoptosis was analyzed by flow cytometry. The activity of CaN in the cardiomyocytes was detected. The protein expression of CaN was determined by Western blotting. RESULTS：Compared with control group, the apoptosis of the cardiomyocytes in CaN group was significantly increased. Compared with PQS+H/R group, the cell apoptosis, the expression of Bcl-2 and Bax, the activity of CaN and its protein expression in FK506 group were not significantly different. CONCLUSION：Inhibition of CaN activity reduces the H/R injury in cardiomyocytes. However, the mechanism of PQS protecting cardiomyocytes from H/R injury may not be associated with the CaN signaling pathway.     

Effect of bim silencing by siRNA on hypoxia-induced apoptosis of rat cardiomyocytes

AIM：To investigate the effect of BH3-only protein Bim (Bcl-2 interacting mediator of cell death) on apoptosis of rat cardiomyocytes induced by hypoxia. METHODS：Rat cardiomyocytes were isolated from infant rats aged 1~3 days and then primarily cultured. The antibody targeting α-actin of striated muscle was used to identify the cardiomyocytes. The siRNAs of bim were transfected into the cardiomyocytes with liposome, and the expression of Bim was determined by Western blotting. The cardiomyocytes were divided into blank control group, hypoxia group, hypoxia+liposome group, hypoxia+negative control siRNA group and hypoxia+bim-siRNA group.The frequency and rhythm of cardiomyocyte beating were observed and recorded under inverted microscope. The activity of lactate dehydrogenase (LDH) in the culture medium was assessed by automatic biochemical analyzer. The viability of the cells was analyzed by MTT assay. The cell apoptotic rate was measured by flow cytometry. The protein expression of Bim, Bax, Bcl-2, p-p38 MAPK and p38 MAPK was detected by Western blotting. RESULTS：Immunohistochemical identification confirmed that the rat cardiomyocytes were successfully cultured. The expression of Bim was obviously inhibited after transfected with bim-siRNAs and the silencing efficiency of bim-siRNA-2 was the highest (86.73%). The frequency of cardiomyocyte beating was slowed down after hypoxia and the rhythm was disordered, while the frequency of beating was obviously increased after silencting the expression of bim. Compared with control group, the LDH in the culture medium was increased (P<0.01), and the viability of the cardiomyocytes was reduced in hypoxia group (P<0.05). The apoptotic rate was increased (P<0.01). After transfection with bim-siRNA, the release of LDH was decreased, and the viability of the cardiomyocytes was increased. The apoptotic rate was decreased. The results of Western blotting showed that hypoxia increased the expression of Bax and p-p38 MAPK (P<0.05), and decreased the expression of Bcl-2 (P<0.01), while transfection with bim-siRNA reduced the effects caused by hypoxia (P<005). These were greatly related to the decrease of apoptosis. However, the expression of p38 MAPK was not changed. CONCLUSION：The apoptosis of cardiomyocytes induced by hypoxia can be inhibited by silencing the expression of bim gene by down-regulation of p-p38 MAPK and Bax expression and up-regulation of Bcl-2 expression.     

Protective role of ornithine decarboxylase (ODC)/polyamines system in the myocardium induced by ischemic preconditioning in rats

AIM: To explore the protective role of ornithine decarboxylase (ODC)/polyamines system in the myocardium induced by ischemic preconditioning in rats.METHODS: The experiment model of simulating myocardial ischemia-reperfusion was replicated by Langendorff perfused rat heart. The hearts were randomly divided into six groups: control group, ischemic-reperfusion group (IR), weak ischemic preconditioning group (IPCw), strong ischemic preconditioning groups (IPCs) and inhibitor groups (DF-EG-IPCw and DF-EG-IPCs). The expression of ODC was quantified by Western blotting analysis. The contents of polyamines (putrescine, spermidine, spermine) in cardiac tissue were detected with high performance liquid chromatography. The hemodynamics was obtained using the PowerLab 8/SP TM data acquisition system. The infarct size was measured using triphenyltetrazolium chloride (TTC) staining and the apoptosis cardiomyocytes were observed under optic microscope after TUNEL method treatment. RESULTS: In contrast with control group, in IR group the putrescine contents increased, the expression of ODC was down-regulated, the contents of spermine and the total polyamine pool decreased (P<0.05). At the same time, the cardiac function declined, with an increase in myocardium infarct size and the apoptosis rate of cardiomyocytes (P<0.05). When compared with IR group in terms of LVDP, HR and CF, both IPCw and IPCs groups had significant improvements in cardiac functions (P<0.05). These two groups also had smaller myocardium infarct size (P<0.01) and apoptosis rate of cardiomyocytes (P<0.01). When compared with IR group, the expression of ODC, the contents of spermine and the total polyamine pool increased in both IPCw (P<0.05) and IPCs groups (P<0.01), but the putrescine contents declined. In the respective inhibitor groups of the weak and ischemic preconditioning, the expression of ODC and the levels of putrescine, spermidine, spermine and the total polyamine pool decreased remarkably (DF-EG-IPCw vs IPCw, P<0.05; DF-EG-IPCs vs IPCs, P<0.01), while the myocardium infarct size and apoptosis rate of cardiomyocyte were relatively bigger in both inhibitor groups (P<0.05). Also, the heart function decreased significantly in terms of LVDP, HR and CF compared to their matched ischemic preconditioning group (P<0.05).CONCLUSION: Ischemic preconditioning significantly up-regulates the ODC / polyamines system in Langendorff perfused rat hearts and provides protective effects on myocardium with ischemia/reperfusion injury. Inhibition of bio-synthesis of polyamine abolishes the cardiac function improvement and the decreases the infarct size and apoptosis rate induced by ischemic preconditioning. It reveals that the ornithine decarboxylase (ODC) /polyamines system may be involved in the protection of myocardium induced by IPC in rats.     

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AIM: To investigate the contribution of angiotensin-converting enzyme inhibitor (ACEI) to the regulation of calpain system in infarcted myocardium. METHODS: Rat myocardial infarction (MI) model was established by permanent ligation of the left coronary artery. The treatment with the ACEI inhibitor rampril (1 mg·kg-1·d-1) was started 7 days prior to surgery. On day 1, 3, 7 and 14 after MI, protein levels of calpainⅠ, Ⅱ and calpastatin were determined in left ventricular free wall (LVFW), interventricular septum (IS) and right ventricule. RESULTS: CalpainⅠprotein level was increased in IS 14 d post MI, whereas the protein level of calpainⅡ was maximally increased in LVFW 3 d post MI. Rampril decreased protein up-regulation of calpainⅠ and Ⅱ, and reduced infarct size and interstitial fibrosis. Calpastatin protein expression was not affected by ACEI. CONCLUSIONS: CalpainⅠ is involved in cardiac remodelling in the late and calpainⅡ contributes to cardiac tissue damage in the early phase of MI. The heart protective effect of ACEI may be related to the inhibition of calpain system in the pathogenesis of myocardial infarction.     

Role of endoplasmic reticulum stress in Bim-mediated cardiomyocyte apoptosis induced by hypoxia

AIM:To investigate the role of endoplasmic reticulum stress (ERS) in the process of Bim-mediated cardiomyocyte apoptosis induced by hypoxia. METHODS:Cardiomyocytes were isolated from neonatal Sprague-Dawley rats aged 1~3 days, and primarily cultured in vitro. The antibody targeting α-striated muscle actin was used to identify the cardiomyocytes. The siRNAs targeting bim were transfected into cardiomyocytes with liposome, followed by detecting the expression of Bim by Western blotting. Cardiomyocytes were divided into five groups: blank control group, hypoxia group, hypoxia+liposome group, hypoxia+negative control siRNA group and hypoxia+Bim-siRNA group. The cell viability was determined by MTT assay, and the cell apoptotic rate and the intracellular calcium concentration were measured by flow cytometry. The protein expression of caspase-12 and inositol 1,4,5-triphosphate (IP3) was detected by Western blotting. RESULTS:Immunohistochemical identification confirmed that rat cardiomyocytes were successfully cultured. Green fluorescence was observed in the cells transfected with negative control siRNA under fluorescence microscope. The expression of Bim was obviously inhibited after transfected with Bim-siRNAs and the silencing efficiency of Bim-siRNA-2 was the highest (86.73%). Compared with blank control group, the viability of cardiomyocytes in hypoxia group was significantly reduced (P<005). Compared with hypoxia+negative control siRNA group, the viability of cardiomyocytes in hypoxia+Bim-siRNA group was significantly increased (P<005). The apoptotic rate and the intracellular calcium concentration of cardiomyocytes were obviously increased in hypoxia group (P<0.01), and were both decreased after bim silencing (P<005 or P<0.01). The expression of caspase-12 and IP3 was up-regulated in hypoxia group (P<005), and was down-regulated after bim silencing (P<005 or P<0.01). CONCLUSION: Cardiomyocyte apoptosis induced by hypoxia can be inhibited by silencing the expression of bim gene. Caspase-12 and IP3, as markers of ERS, may participate in the process of Bim-mediated cardiomyocyte apoptosis induced by hypoxia.     

miR-499 attenuates myocardial injury in rats by targeting PTEN

AIM:To investigate the effect of microRNA-499 (miR-499) on myocardial injury in rats and its mechanism. METHODS:The rat model of ischemia-reperfusion (I/R) injury was established and the myocardial cells were primarily cultured. The expression level of miR-499 was detected by RT-qPCR. After hydrogen peroxide stimulation, CCK-8 assay and LDH kit were used to detect the viability and LDH release of the cells transfected with miR-499 mimic and siR-PTEN. The targeting relationship between miR-499 and PTEN was predicted by TargetScan and confirmed by luciferase test. RT-qPCR and Western blot were used to detect the effect of miR-499 mimic and inhibitor on the mRNA and protein expression levels of PTEN. After miR-499 inhibitor and siR-PTEN were co-transfected into the cells, CCK-8 assay and LDH kit were used to detect the viability and LDH release of the myocardial cells induced by hydrogen peroxide. RESULTS:The expression level of miR-499 in the I/R rats was increased rapidly, and then was decreased gradually in a time-dependent manner (P<0.05). miR-499 mimic and siR-PTEN significantly promoted the viability and decreased the LDH release of cardiomyocytes induced by hydrogen peroxide (P<0.05). miR-499 and PTEN had a targeting relationship. The expression of PTEN was significantly down-regulated by miR-499 mimic, and up-regulated by miR-499 inhibitor (P<0.05). Transfection with siR-PTEN reversed the inhibitory effect of miR-499 inhibitor on the cells. CONCLUSION:miR-499 attenuates the myocardial injury in rats by targeting PTEN.     

MicroRNA profile analysis of ischemic myocardial tissues from rats with acute myocardial infarction

AIM：To identify differentially expressed microRNAs (miRNAs) in ischemic myocardial tissues from the rats with acute myocardial infarction (AMI) by miRNA array technique, and to predict their targets and analyze their functions using bioinformatics. METHODS：The rat models of AMI (n=3) were prepared by ligaturing the left anterior descending coronary artery (LAD) of Wistar rats. Electrocardiogram and blood pressure were detected during the operation, and the myocardial infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Ischemic myocardial tissues were isolated from the infarct area 4 h after ischemia. The same procedure in sham group (n=3) was performed except for ligaturing LAD. Total RNA was extracted from ischemic and normal myocardial tissues. miRNA was isolated from total RNA, labeled with Cy3 and hybridized on miRNA array. Real-time PCR was applied to verify the reliability of miRNA array results. The targets of differentially expressed miRNAs were predicted and their functions were analyzed by bioinformatics. RESULTS：Rat model of AMI was successfully prepared and verified by electrocardiogram detection, blood pressure measurement and pathological observation. Compared with sham group, microarray screening showed that total 11 AMI-related miRNAs were selected, including 6 up-regulated and 5 down-regulated. Three of them (rno-miR-181c, rno-miR-146b and rno-miR-208) were related to the cardiovascular functions, while the functions of the others (rno-miR-672*, rno-miR-743b, rno-miR-128, rno-miR-138-1*, rno-miR-336, rno-miR-138-2*, rno-miR-325-3p and rno-miR-3572) were unknown and might be novel AMI-related biomarkers. Parts of the miRNA targets were also related to the cardiovascular functions. CONCLUSION：Differentially expressed miRNAs in AMI rats may serve as novel biomarkers for diagnosis of AMI and potential targets for treatment of AMI.     

Microparticles derived from bone marrow mesenchymal stem cells promote angiogenesis in rat myocardial infarction model

AIM: To observe the effects of microparticles derived from bone marrow mesenchymal stem cells (MSC-MPs) on angiogenesis and cardiac function in a rat myocardial infarction model. METHODS: MSCs were obtained from Sprague-Dawley rats. MSCs were treated under serum-free condition in hypoxia for 72 h, and the microparticles were isolated from the supernatants. The phenotypic profile of MSC-MPs was determined by bead-based flow cytometry and the morphology was observed under a transmission electron microscope. The rat myocardial infarction model was established. The cardiac function was evaluated by echocardiography after the intramyocardial injection of MSC-MPs. The myocardial infarct size was observed by Masson staining. The blood vessel density in the peri-infarcted area was measured using immunohistochemical staining for von Willebrand factor and α-smooth muscle actin. The expression of vascular endothelial growth factor (VEGF) was analyzed by real-time PCR. RESULTS: Apoptotic MSCs released a large quantity of microparticles which were phenotypically similar to the parent MSCs and 100~1 000 nm in diameter. The cardiac functions of myocardial infarction rat model were improved at 7 d and 28 d after intramyocardial injection of MSC-MPs compared with control group. The myocardial infarct size was reduced and angiogenesis was promoted significantly in the infarcted heart injected with MSC-MPs 28 d after treatment. MSC-MPs treatment also increased the expression level of VEGF within 7 d.CONCLUSION: MSC-MPs protect cardiac tissue from ischemic injury and improve cardiac function by promoting angiogenesis after myocardial infarction.     

Aldehyde dehydrogenase 2 prevents rat cardiomyocytes from apoptosis induced by hypoxia

AIM: To observe the effect of hypoxia on cardiaomyocytes apoptosis and the role of ALDH2 in the process. METHODS: Cultured cardiomyocytes of neonatal rats were used. Hypoxia was imposed to the cardiomyocytes with or without daidzin pretreatment. ALDH2 activity was measured by the method of acetaldehyde metabolism. Apoptosis was measured by Hoechest 33324 staining, fluorescence activated cell sorting (FACS) and the DeadEndTM fluorometric TUNEL system. RESULTS: ALHD2 enzyme activity in myocytes was inhibited by daidzin (24 h, 60 μmol/L) without induction of apoptosis. When exposed to hypoxia, however, the apoptisis was significantly increased in the cells pretreated with daidzin compared to those without the pretreatment. CONCLUSION: The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosis following hypoxia, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury.     

Effect of cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor gene transferred neonatal cardiomyocytes

AIM:To observe the secretion of vascular endothelial growth factor (VEGF) when adenovirus induced VEGF165 (AdVEGF165) gene transferred into neonatal cardiomyocytes in vitro,and to investigate the impact on heart function after transplanted the transferred cardiomyocytes into infarct myocardium in rats.METHODS:Neonatal cardiomyocytes were cultured in vitro and labeled with BrdU,then transferred by AdVEGF165.ELISA was applied to assay the expression and secretion of VEGF.Wistar rats,in which left descending branch of coronary artery was ligated,were randomly divided into four groups and transplanted into MI area with transferred cardiomyocytes (group Ⅰ),untransferred cardiomyocytes (group Ⅱ),AdVEGF165 (group Ⅲ) and culture medium (group Ⅳ),respectively.The echocardiograph was applied to evaluate the heart function before and after cell transplantation.Then the rats were executed and the hearts were harvested for histological (hematoxylin-eosin) and immunohistological (anti-BrdU dyeing) examinations.The vessels were also counted in injected area.RESULTS:ELISA indicated that the expression and secretion of VEGF in groupⅠwere higher than those in the rest (P<0.01).Echocardiograph revealed that the improvement of ejection fraction (EF) in groupⅠwas greater than that in other groups (P<0.01).Immunohistological study showed that the implanted cardiomyocytes survived in MI area.More blood vessels in groupⅠthan other groups were observed (P<0.01) by vessel-counting.CONCLUSIONS:AdVEGF165 gene transfer increased the VEGF-production,accelerated neovascularization in MI area and increased the blood flow.The method makes favor for the survival of implanted cells and greatly improves the heart function.     

Prostaglandin E1 protects heart after myocardial infarction by inhibiting endoplasmic reticulum stress in rats

AIM To investigate the effect of prostaglandin E1 (PGE1) on heart after myocardial infarction (MI) in rats and its related molecular mechanism. METHODS Fifty male SD rats were divided into sham group, model group and model+PGE1 group. The MI rat model was established by ligation of left anterior descending coronary artery. Cardiac function in the rats was detected by echocardiogaphy. The myocardial histomorphologic changes were evaluated by HE and Masson staining. The MI area was measured by TTC staining. The cardiomyocyte death was detected by TUNEL staining. The protein levels of endoplasmic reticulum stress (ERS)-related factors glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and caspase-12, and apoptosis-related factors Bcl-2, Bax and cleaved caspase-3 were determined by immunofluorescence staining and Western blot. RESULTS Compared with sham group, the cardiac function in model group was decreased, with significant myocardial pathological changes. The MI area was enlarged, and the death of cardiomyocytes was promoted. The protein levels of GRP78, CHOP, caspase-12, Bax and cleaved caspase-3 in the myocardial tissues were significantly increased, while Bcl-2 was decreased (P<0.01). Compared with model group, the cardiac function in model+PGE1 group was significantly improved, and the myocardial pathological damage was significantlty attenuated. The MI area and myocardial cell death were significantly reduced. The protein levels of GRP78, CHOP, caspase-12, Bax and cleaved caspase-3 in the myocardial tissues were significantly decreased, while Bcl-2 was increased (P<0.01). CONCLUSION PGE1 reduces collagen deposition and inflammation, and improves cardiac function by reducing ERS level, thus protecting cardiomyocytes from MI damage.     

Protective role of 14-3-3γ in burn and LPS-induced rat myocardial injury

AIM: To study the protective role of 14-3-3γ in burn or LPS-induced myocardial injury. METHODS: The rat model of burn or LPS-induced injury was established. The heart functions and 14-3-3γ protein expression were detected 3 h, 6 h, 12 h and 24 h after treatment. Primary neonatal rat cardiomyocytes were used in vitro. pFLAG-14-3-3γ plasmid was constructed and transfected into the cardiomyocytes 24 h before LPS-induced injury. The injury in the cardiomyocytes was evaluated by measuring the cell viability and the level of lactate dehydrogenase(LDH). Apoptosis of cardiomyocytes was detected by flow cytometry. Opening of mitochondrial permeability transition pore (mPTP) was also determined by Ca²⁺-induced swelling of isolated myocardial mitochondria. RESULTS: The expression of 14-3-3γ was elevated following the burn or LPS-induced myocardial injury in vivo. In vitro, transfection with pFLAG-14-3-3γ plasmid in to the cardiomyocytes significantly protected against LPS-induced injury. Compared with the cardiomyocytes without transfection with pFLAG-14-3-3γ plasmid, higher cell viability rate and lower LDH release, cell apoptosis and mPTP opening were observed in the cardiomyocytes transfected with pFLAG-14-3-3γ plasmid. CONCLUSION: The 14-3-3γ protein protects the heart against burn or LPS-induced injury by inhibiting the mPTP opening.     

Role of TGF-β1/Smad signaling in angiotensin Ⅱ mediated down-regulation of connexin 43

AIM: To analyze the alterations of angiotensin Ⅱ (Ang Ⅱ), connexin 43 (Cx43), angiotenisin Ⅱ receptor type 1 (AT1) and signaling molecules in the TGF-β1/Smad pathway in different regions of the left ventricular heart tissue for exploring whether Ang Ⅱ regulates Cx43 expression via the TGF-β1/Smad signaling pathway in myocardial infarction (MI) rats. METHODS: MI was induced in 20 male Sprague-Dawley rats by the left anterior descending coronary artery ligation. The rats were then randomized into 2 groups. In the losartan group, 20 mg·kg^-1·d^-1 of losartan were administered for 2 weeks. Heart functions were assessed after surgery and 2 weeks later again following the above treatments. All the rats were sacrificed and relevant molecules, including Ang Ⅱ, AT1, and Cx43 were determined thereafter in diffe-rent areas of the left ventricle. TGF-β1 and its downstream signaling molecules, including Smad 2, Smad 3 and Smad 7, were also detected. RESULTS: In losartan group, both left ventricular internal dimension diastole (LVIDd) and left ventricular internal dimension systole (LVIDs) were smaller, with diminished interventricular septal thickness (IVSd) and left ventricular posterior wall depth (LVPWd) and distinct improvement of left ventricular ejection fraction (LVEF) (P<0.05). Losartan therapy exhibited a reduction of Ang Ⅱ in the infarct zone and the border zone in the cardiac tissues. AT1 was obviously attenuated in the infarct zone with an enhanced expression of Cx43, which was also elevated in the border zone and none infarct zone. TGF-β1, Smad 2 and Smad 3 were decreased in different zones of the left ventricle, while Smad 7, in contrary to the above factors, presented a converse alteration.CONCLUSION: The activation of Ang Ⅱ provokes downregulation of Cx43 through TGF-β1/Smad signaling pathway in MI rats.     

Hypoxia promotes apoptosis of neural stem cells and down-regulates miR-26a

AIM: To investigate the effect of cobalt chloride (CoCl₂) on the apoptosis of neural stem cells (NSCs) and the expression of microRNA-26a (miR-26a) in vitro, and to explore the mechanisms of NSC apoptosis induced by CoCl₂. METHODS: NSCs were exposed to CoCl₂ at different doses (200~600 μmol/L) for 24 h. The cell viability and apoptosis were measured by CCK-8 assay and TUNEL method. The expression of miR-26a-3p, miR-26a-5p, GSK-3β, caspase-3, Bcl-2 and Bax was examined by real-time PCR. The protein levels of Bcl-2 and Bax were detected by Western blotting. RESULTS: The cell viability was inhibited and the apoptosis of NSCs was increased significantly by CoCl₂ in a dose-dependent manner (P<0.05). CoCl₂ at concentration of 400 μmol/L for 24 h was used to induce apoptosis and the expression of miR-26a was down-regulated compared with control (P<0.05). Exposure to CoCl₂ at concentration of 400 μmol/L up-regulated the expression of GSK-3β, caspase-3 and Bax, down-regulated the expression of Bcl-2 and Bcl-2/Bax (P<0.05). CONCLUSION: CoCl₂ at concentration of 400 μmol/L induces the apoptosis of NSCs obviously. CoCl₂ may induce the NSC apoptosis by mitochondrial apoptotic pathway. Declining miR-26a may be related to NSC apoptosis.