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Aim To investigate the protective roles of sonic hedgehog( Shh) signaling pathway in hypoxia-in-duced DNA damage with the neonatal rat cardiomyo-cytes. Methods The hypoxia model on neonatal car-diomyocytes was established with one to two days old Sprague Dawley rats by deprivation of oxygen and glu-cose ( OGD) . After pretreated with Shh pathway ago-nist SAG1.3 or antagonist GANT61, the survival rates of cardiomyocytes were assayed by MTT after OGD 6 hours or 12 hours. The protein levels of Shh pathway, phosphorylated histone H2AX at serine 139 (γH2AX), phosphorylated ATM (p-ATM), phospho-rylated p53 ( p-p53 ) , cleaved-caspase-3, Bcl-2 and Bax were detected by Western blot. The γH2AX foci was detected by immunofluorescence. Results Com-pared to control group, the protein expression of γH2AX, p-ATM, cleaved-caspase-3, p-p53 in OGD cardiomyocytes significantly increased, and Bcl-2/Bax ratio proportionally decreased. Particularly, the ex-pression of γH2AX, p-ATM was highest at OGD 6 h, and then gradually declined after OGD 12 h. After SAG1.3 pretreatment, the expression of γH2AX, p-ATM, cleaved-caspase-3 and p-p53 dramatically de-creased and the Bcl2/Bax ratio increased in OGD 6 h or OGD 12 h cardiomyocytes. On the contrary, in GANT61 pretreatment group, the expression of γH2AX, p-ATM, cleaved-caspase-3 and p-p53 signifi-cantly increased and the Bcl-2/Bax ratio decreased compared to the OGD 6 h or OGD 12 h cardiomyo-cytes. Conclusion The activation of Shh pathway protects cardiomyocytes against hypoxia-induced apop-tosis through inhibition of DNA damage.
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Objective To explore the effects of miRNA-204 targeted LC3B expression on Ang Ⅱ induced cardiomyocytes hypertrophy.Methods The primary neonatal rat cardiomyocytes served as the research objects and divided into the control group,AngⅡ group,combination-treated group 1 (cardiomyocytes were given Ang Ⅱ stimulation,meanwhile infected by negative control lentivirus vector),combination-treated group 2 (cardiomyocytes were given Ang Ⅱ stimulation,meanwhile infected by lentivirus carrying miRNA-204 overexpression vector) according to different treatments.About 48 h to 72 h after intervention treatment,the cardiomyocyte hypertrophy change was detected by confocal microscopy,the expression of miRNA-204 was analyzed by real time PCR,the protein expression of LC3B was measured by Western blot and targeted gene of miRNA-204 was demonstrated by dual-luciferase reporter assay system.Results Compared with the control group,the cardiomyocyte relative surface area in the Ang Ⅱ group was significantly enlarged,the protein expression of LC3B was significantly increased,the expression of miRNA-204 was upregulated,the differences were statistically significant (P＜0.05).Whereas comparing the combination-treated group 1 with combination-treated group 2,the protein expression of LC3B in the latter was down-regulated and the cell area was reduced (P＜0.05).The further luciferase activity report gene experiment results suggested that miRNA-204 was able to bind to LC3B 3'-UTR and decreased the luciferase activities (P＜0.05),but not to bind its mutated fragment for inactivating luciferase activity(P＞0.05).Conclusion miRNA-204 is able to inhibit Ang Ⅱ induced cardiomyocytes hypertrophy,its action is realized by targeting the expression of LC3B.
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AIM: To prepare and purify the polyclonal antibodies against human myofibrillogenesis regulator 1 (hMR-1), then to characterize the purity, titer, specificity and the availability.METHODS: Two polypeptides named peptide 1 and 2 were synthesized based on the bioinformatics analysis of the sequence of hMR-1 by using software TMHMM and DNAStar, then coupled with keyhole limpet hemocyanin (KLH) for immunization. These peptides for immunization were mixed and injected into New Zealand rabbits to prepare antibodies specifically against hMR-1. ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, antibodies were identified by Western blotting and immunocytofluorescent assays. Applications of the antibodies on neonatal rat cardiomyocytes were also employed.RESULTS: (1)The titers of antibodies were 1:10~5. In WB assay, a specific 17kD band was detected, corresponding to the predicted molecular weight of hMR-1; the positive fluorescent signals were distinct. (2)On the neonatal rat cardiomyocytes model, we observed a peri-nucleus location. The fluorescent signal of hMR-1 overexpression group was much stronger than that in vector control and normal control groups.CONCLUSION: All these results indicate that the antibodies obtained from poly peptides mixture immunization have either human original or rat original antigens. The antibody is available for using in Western blotting or immunofluorescent assays.
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Objective To investigate the effect and mechanism of different high concentrations glucose(Glu) on the expression of hypoxia-inducible factor 1α(HIF-1α) in cultured neonatal rat eardiomyocytes under hypoxia and non-hypoxia conditions. Methods Neonatal rat cardiomyocytes were cultured for 6 hours under different conditions and were divided into 6 groups:①Negative control group (5.5 mmol/L Glu) ; ②hypoxia mimicking cobalt chloride(Cocl_2) group(5.5 mmol/L Glu + 400 μmol/L cocl_2 ) ;③Different high concentrations Glu groups: (11.1 mmol/L, 22.2 mmol/L, 33.3 mmol/L Glu);④cocl_2 +different high concentrations Glu groups(11.1 mmol/L Glu +400 μmol/L cocl_2,22.2 mmol/L Glu +400 μmol/L cocl_2, 33.3 mmol/L Glu+400 μmol/L cocl_2);⑤High concentrations Glu + antioxidant α-tocopherol group(33.3 mmol/L Glu + 100 μmol/L α-tocopherol) ; ⑥ High concentrations Glu+antioxidant α-tocopherol+cocl_2 group(33.3 mmol/L Glu+400 μmol/L cocl_2+100 μmol/L α-tocopherol). The effect of high concentrations Glu, cocl_2 and high concentrations Glu plus cocl_2 on the expression of HIF- 1α mRNA and protein in cultured neonatal rat cardiomyocytes, as well as the effect of high concentrations Glu plus antioxidant α-tocopherol, high Glu concentrations plus cocl_2 and antioxidant α-tocopherol on the expression of HIF- 1α mRNA and protein were observed. Results 1. Compared with negative control group(5.5 mmol/L Glu), the 'expression of HIF- 1α was increased under cocl_2 mimicked hypoxia(5.5 mmol/L Glu+400 μmol/L cocl_2, 11.1 mmol/L Glu+400 μmol/L cocl_2 ,22.2 mmol/L Glu+400 μmol/L cocl_2,33.3 mmol/L Glu+400 μmol/L cocl_2). 2. The expression of HIF- 1α was increased gradually after the increasing of Glu concentrations(5.5 mmol/L, 11.1 mmol/L, 22.2 mmol/L and 33.3 mmol/L Glu).3.The expression of HIF-1α was decreased gradually after the increasing of Glu concentrations under certain cocl_2 plus different high concentrations Glu (5.5 mmol/L Glu+400 μmol/L cocl_2, 11.1 mmol/L Glu+400 μmol/L cocl_2, 22.2 mmol/L Glu+400 μmol/L cocl_2,33.3 mmol/L Glu+400 μmol/L cocl_2). 4. Under high concentration Glu plus antioxidant α-tocopherol (33.3 mmol/L Glu+100 μmol/L α-tocopherol) ,the expression of HIF- 1α was less increased than the same high Glu concentration(33.3 mmol/L Glu). 5. Under high Glu concentration plus cocl_2 (33.3 mmol/L Glu+400 μmol/L cocl_2), the expression of HIF- 1α increased less than that of the same high Glu concentration plus antioxidant α-tocopherol and cocl_2 (33.3 mmol/L Glu+400 μmol/L Cocl_2+ 100 μmol/L α-tocopherol). Conclusions High glucose increases the expression of HIF-1α under non-hypoxia, but blunts the expression of HIF-1α under hypoxia in cultured neonatal rat cardiomyocytes. Some mechanisms such as ROS (reactive oxygen species) signal transduction system and oxidative stress may be involved in it.
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The effects of cardiomyocyte grafting on left ventricular (LV) remodeling and function in rats with chronic myocardial infarction were evaluated using high-frequency ultrasound. Chronic myocardial infarction was induced in 50 Wister rats by ligating the left anterior descending artery. They were randomized into two groups: a trial group that received neonatal rat cardiomyocyte trans- plantation (n=25) and a control group which were given intramyocardial injection of culture medium (n=25). The left ventricular (LV) geometry and function were evaluated by high-frequency ultrasound before and 4 weeks after the cell transplantation. After the final evaluation, all rats were sacrificed for histological study. The results showed that 4 weeks after the cell transplantation, as compared with the control group, the LV end-systolic dimension, end-diastolic dimension, end-systolic volume and end-diastolic volume were significantly decreased and the LV anterior wall end-diastolic thickness, LV ejection fraction and fractional shortening were significantly increased in the trial group (P<0.01). Histological study showed that transplanted neonatal rat cardiomyocytes were found in all host hearts and identified by Brdu staining. It was suggested that transplantation of neonatal rat cardiomyocytes can reverse cardiac remodeling and improve heart function in chronic myocardial infarction rats. High-frequency ultrasound can be used as a reliable technique for the non-invasive evaluation of the effect of cardiomyocyte transplantation.
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Aim To investigate the protection effect of icariin on isoproterenol induced neonatal rat cardiomyocytes injury.Methods Isolation of cardiomyocytes from Sprague-Dawley rats aging 1～3 d were performed.Cardiomyocytes were grown in DMEM culture medium containing 15% fetal bovine serum.The cell density was adjusted to 1?105～5?105 cells?L-1,and the suspension was pipetted into each well of culture plates.Cells were incubated at 37 ℃ in 5% CO_2 atmosphere for 72 h.Cultured rat cardiomyocytes were divided into different groups:control,0.5 mmol?L-1 isoproterenol(model),and therapeutic groups with 0.1,1 and 10 ?mol?L-1 icariin.Cell viability was analyzed using MTT assay.The potential of mitochondrial membrane was determined by laser scanning confocal microscope.Apoptotic cells were detected by using flow cytometric analysis.Results Compared with the control group,the cell activity was lower in model group(P
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AIM:To observe the effects of adiponectin on injury of the 3-4 d SD rat cardiomyocytes induced by the intervention of H2O2.METHODS:Primary cardiomyocytes were obtained from neonatal rat and were cultured by enzymatic digestion methods.The molecular marker was observed by ?-actin immunocytochemistry.Primary cultured 3-4 d cells were used in experiment,and the injury model was established by H2O2,and adiponectin and Ara-A were used for pre-treatment before cell culture.The morphological change of cardiomyocytes was observed under electron microscope.The contents of LDH,MDA and the activity of SOD were measured.The apoptosis of cardiomyocytes was detected by agarose gel electrophoresis and Annexin V/PI staining with flow cytometry.RESULTS:Adiponectin pretreatment significantly decreased the release of LDH(P