Wu Yanqing

MicroRNA-223 prevents cardiomyocyte hypertrophy by targeting cardiac troponin I-interacting kinase

Objectives Our study was designed to investigate the role of microRNA-223 (miR-223) and its direct target gene, cardiac troponin I-interacting kinase (TNNI3K), in regulating cardiomyocyte hypertrophy. Methodology Neonatal rat cardiomyocytes (CMs) were cultured from one to two days old Sprague–Dawley rats. Cardiomyocyte hypertrophy was induced by endothelin-1 (ET-1). Expression of miR-223 in CMs was detected by real-time PCR. miR-223 mimics transfection was performed to achieve overexpression of miR-223 in CMs. Cell size was measured via surface area calculation under fluorescence microscopy after anti-α-actinin staining. Expression levels of ANP, α-actinin, Myh6, Myh7, as cardiac hypertrophy related marker genes, were detected by RT-PCR. The expression of TNNI3K protein was analysed by western blot. Luciferase assay was performed to confirm the direct binding of miR-223 to the 3′UTR of TNNI3K mRNA. Results In ET-1 induced hypertrophic CMs, expression of miR-223 was lower than that in normal CMs (Normal CMs: 1.00±0.08 vs Hypertrophic CMs: 0.62±0.16, p<0.05). Under stimulation of ET-1, miR-223 overexpressed CMs showed alleviated hypertrophic phenomenon, which characterised by less cell surface area (miR-223 group: 2590±781 mm2 vs ET-1 only group: 4680±1040 mm2, p<0.01) and lower expression of ANP, α-actinin, Myh6, Myh7, when compared with ET-1 stimulation only CMs. In miR-223 overexpressed CMs, the expression of TNNI3K protein was significantly decreased (miR-223 group: 0.39±0.05 vs Control: 0.03±0.01). Co-transfection of a miR-223 expression vector with pMIR-TNNI3K led to the reduced activity of luciferase in a dual-luciferase reporter gene assay, suggesting that TNNI3K is a direct target gene of miR-223. Conclusion All these results suggest that TNNI3K, a novel cardiac-specific kinase gene, is a direct target of miR-223. miR-223 plays a important role as suppressor in cardiomyocyte hypertrophy and could be used in clinical treatment of hypertrophy in future.

GW24-e2991 Protective effect of cardiotrophin-1 in preventing acute cardiomyocyte hypoxia- reoxygeonation injury mediated by supressor of cytokine signalling 1--SOCS1/stat3 pathway

Objectives To investigate the signalling pathways supressor of cytokine signalling 1 (SOCS1) how to involve in the protective effect of CT-1 in preventing cardiomyocyte I/R injury from cellular and molecular aspects. Methods Cardiomyocytes from the hearts of 1∼3-day-old neonatal rats (Sprague-Dawley) were prepared by a modification of a previously published protocol. There were seven groups in the experiment: 1) control group: cultivated with DMEM for 6h; 2) hypoxia-reoxygeonation group: 3) CT-1 group; 4) ASODN group1 and group 2: antisense oligonucleotides group (two antisense SOCS1 groups) : 5) SODN group (sense oligonucleotides SOCS1 group); 6) ScODN group (scrambled oligonucleotides SOCS1 group). Myocytes survival rate was evaluated by MTS method, apoptosis, mitochondrial permeability transition pore (Δψm) and reactive oxygen species (ROS) were detected by flow cytometer. SOCS1 and STAT3 protein by western blotting. Results Cardiomyocyte apoptosis and ROS increased markedly after hypoxia/reoxygenation, but cardiomyocyte survival rate and the level of Δψm decreased significantly. With CT-1 intervention, cardiomyocyte survival rate increased markedly (87%), apoptosis and ROS reduced significantly Δψm was lower and the ROS was higher than those in CT-1 group after giving antisense SOCS1 infection, but it was similar to that in CT-1 group after sense SOCS1 or scrambled SOCS1 infection. After hypoxia-reoxygeonation, Expression of SOCS1 mRNA and SOCS1 protein increased somewhat comparing with control group, but there no significant difference. However,expression of both SOCS1 mRNA and SOCS1 protein in CT-1 group increased apparently comparing with hypoxia-reoxygeonation group. Phosphorylated STAT3 increased obviously after processing with SOCS1 antisense oligonucleotides. Conclusions Supressor of cytokine signalling 1--SOCS1/STAT3 pathway may mediate the protection of CT-1 in preventing acute cardiomyocyte hypoxia- reoxygeonation injury.

GW24-e2216 HSP22 expression in the aorta of rat fed a high-fat diet and the effect of statins intervention

Objectives Study the expression of Heat shock protein 22 in the aortic hyperlipidemia rat and the effect of atorvastain intervention. Methods 12 female SD rats were divided into normal group (n = 4) and model group (n = 8). Normal group was fed rodent chow and model group was fed high-fat diets. All of the rats in the model group were given intraperitoneal injections 0f vitamin D3 with total dose of 700000U/kg during the first three days. The serum lipids were detected after 10 weeks feeding in order to identify hyperlipidemia model. After the formation of hyperlipidemia,the model rats were divided into hyperlipidemia control (n = 4) and statins intervention (n = 4) two groups. Statins intervention group was given storvastain gavage with the dosage of 20mg/kg and fed the high-fat diets daily. Hyperlipidemia control group was still fed high-fat diets. After 21-weeks,all the rats were collected blood samples and aorta. The aorta morphology was observed by HE staining, the expression of HSP22 and TNF-α in the aortic vascular wall were detected by immunohistochemical. Results High-fat diets fed for 10 weeks typical hypercholesterolemia was set up successfully. After the experiment the thoracic and abdominal aorta of the hyperlipidemia control and statins intervention group were manifested exrensive calcification in the media. Chondroid cells proliferation were found in the tissue slice. No significant changes in the normal group. The expression of HSP22 were negative in the normal group,while positive in the hyperlipidemia controlgroup and statins intervention group. The mean density of HSP22 postive particles was lower in the statins intervention group compared to hyperlipidemia control group (0.219 ± 0.090 vs 0.377 ± 0.094, P<0.05). The expression of TNF-α were negative in the normal group, while positive in the hyperlipidemia control group and statins intervention group. There were no significant difference in the mean density of the hyperlipidemia control group and statins intervention group (P>0.05). Conclusions The high-fat diets combine vitamin D3 could slowly induce hyperlipidemia and arterosclerosis rat model. Hyperlipidemia could increase the expression of HSP22 in the rat aorta. HSP22 may play a protective role in the hyperlipidemia induced artery lesion. Atorvastain could decrease the expression of HSP22 and the mechanism may be partially related to the anti-inflammatory effect.

ASSA13-03-52 Heat Shock Protein 22 Expression in Human Umbilical Vascular Endothelial Cells with Hyperlipemia

Objective To observe heat shock protein 22 (HSP22) gene eukaryotic expression vector pEGFP-N1 transfected HSP22 expression levels in human umbilical vein endothelial cells in hyperlipidemia environment. Methods HSP22 cDNA was obtained from MCF-7 by reverse transcription-polymerase chain reaction (RT-PCR) and linked to pEGFP-N1, and then recombinant plasmid pEGFP-N1/HSP22 was transformed into DH5α. Positive clones were identified by double enzyme digestion and sequencing. Recombinant plasmid pEGFP-N1/ HSP22 was transfected into HUVECs with liposome. The cell model of endothelial cells (EC) hyperlipemia was established to simulate environment of hyperlipemia injury. To observe heat shock protein 22 (HSP22) gene eukaryotic expression vector pEGFP-N1 transfected HSP22 expression levels in human umbilical vein endothelial cells by hyperlipidemia environmental. Results (1) Agarosegel electrophoresis detection showed that HSP22 DNA segment was about 211 bp, which accorded with the expectation. Positive clones were identified by enzyme digestion after HSP22 gene was cloned into pEGFP-N1, and the sequencing results indicated it was correct. (2) RT-PCR methods showed HSP22 mRNA was expressed in transfected HUVECs. Western blot detected a specific protein band of HSP22. (3) HSP22 expression was significantly increased in the high-fat environment. Conclusions The recombinant plasmid pEGFP-N1/HSP22 is successfully constructed and expressed positively with hyperlipemia in HUVECs.

ASSA13-10-16 The Role of Endoplasmic Reticulum Stress in The Injury Induced by Bim of Hypoxic Cardiomyocytes

Objective To investigate the role of endoplasmic reticulum stress in Bim-induced cardiomyocytes injured by hypoxia. Methods Bim-siRNA were transfected transiently into H9C2 cells by lipofectamine 2000. Establish a model of hypoxia (The cells after 48 h transfection, were given a deal with hypoxia for 12 h). The experiments were divided into five groups: Blank Control Group, Hypoxia Group, Hypoxia + Negative Control siRNA Group, Hypoxia + Mock control Group, Hypoxia + Bim-siRNA Group. The cell survival rate was determined by MTT method; the cell apoptosis rate and the change of the calcium concentration in cells were detdemined by flow cytometry; the expression of Bim and the markers of endoplasmic reticulum stress Caspase-12 and In SP3 were assessed by Western Blotting. Results Green fluorescence was observed in the cells transfected of negative control siRNA group though the fluorescence microscope. Compared with the blank control group, The MTT assay determined that the survival rate of H9C2 was decreased (p < 0.05) after the injured by hypoxia. And the results of flow cytometry showed that hypoxia increased cell apoptosis rate (P < 0.01) and the concentration of calcium (p < 0.01), while the transfection of Bim-siRNA reduced the effects caused by hypoxia (P < 0.05 or P < 0.01). Compared with the hypoxia group, the transfection of Bim-siRNA increased the cell survival rate, decreased cell apoptosis rate and the concentration of calcium (p < 0.01 or p < 0.05). While there was no significant difference among Hypoxia Group, Hypoxia + Negative Control siRNA Group and Hypoxia + Mock control Group (p > 0.05). The results of Western blotting showed that the transfection of Bim-siRNA reduced the expression of Bim obviously (p < 0.01); meanwhile, reduced the expression of endoplasmic reticulum stress markers Caspase-12 and InSP3 (p < 0.05 or p < 0.01), and reduced the effects that hypoxia increased the expression of Caspase-12 and InSP3 (p < 0.05 or p < 0.01). Conclusions The down-regulation of the expression of Bim can suppress the apoptosis of H9C2 induced by hypoxia. Its mechanism is associated with the endoplasmic reticulum stress. It is likely to be a new direction for treatment of coronary atherosclerotic heart disease.

ASSA13-15-22 The Inter-Arm Blood Pressure Difference Induced by Transradial Coronary Intervention

Background and Objective Inter-arm blood pressure difference (IAD) indicates artery disease of arm. This study was to investigate whether the transradial coronary intervention (TRI) increases the prevalence of IAD. Methods This study enrolled 154 adult in-patients underwent the first selective TRI, and without radial artery occlusion (RAO) by palpation method after TRI. Bilateral brachial and radial artery BPs were simultaneously measured using two automatic BP measurement devices in the day before (BP0) and one day (BP1d), 7 day (BP7d), 3 month (BP3m) and 6 month (BP6m) after TRI. The difference of the left and right BPs was calculated as the ΔBP l-r and the absolute value of ΔBP l-r equal to or over 10mmHg was recognised as IAD. Results The bilateral brachial and radial SBP0 and DBP0 were similar. The brachial BP1d and BP7d of the catheterized right arm decreased significantly and were significantly lower than those of left arm. At the same time, the rates of sIAD on brachial artery were higher than the baseline (27.3% and 13.0% vs 7.1%, both P < 0.05). Even the brachial artery BP6m was similar to BP0, but sIAD on brachial artery was higher somewhat than baseline (9.1%). Similar results were seen in the bilateral radial artery BP and IAD also. Conclusions TRI can induce BP decrease in the catheterized arm and increase the prevalence of systolic IAD for at least 7 days in both brachial or radial artery sites.

Heme oxygenase-1 downregulate the activity of nuclear factor NF-κB and protect the damage of cardiocyte induced by lipopolysaccharide in rats

Objective To determine whether HO-1 played a important role in the protection of cardiocyte in the lipopolysaccharide-induced acute cardiocyte injury model. Method Cardiocyte was isolated from SD neonate rat, and was enrolled into C group (normal culture), L group (with stimulation of LPS), L+H group (with stimulation of Hemin (inducer of HO-1), followed by LPS), and L+Z group (with stimulation of ZnPP (inhibitor of HO-1), followed by LPS). The levels of LDH, malondialdehyde (MDA), and superoxide dismutase (SOD) were measured. The cell heart rhythm, survival rate and the apoptosis rate were also examined. The expression of nuclear factor κB (NF-κB), HO-1, and tumour necrosis factor α (TNFα) were measured with RT-PCR, Western blotting and flow cytometry. Results The level of LDH and MDA were significantly higher in L group than those in C group (113±15 vs 69±10 U/L, p<0.05, and 1.88±0.36 vs 0.87±0.25 mmol/l, p<0.05, respectively), and decreased in L+H groups (p<0.05). The level of SOD in L, L+H, and L+Z groups was significantly lower than that in control group (p<0.05). The level of SOD in L+H group is higher than that in L group (p<0.05). The rate of apoptosis and cardiocyte heart rhythm increased significantly and survival rate was significantly lower in L, L+H, and L+Z groups than those in C group (p<0.05). The level of HO-1 mRNA was higher in L, L+H, and L+Z groups than that in C group (p<0.01), among which L+H group was the highest. The level of HO-1, TNFα and NF-κB in L, L+H, and L+Z groups was also higher than those in C group (p<0.05), among which the level of HO-1 protein in L+H group was the highest, the level of TNFα and NF-κB was the highest in L+Z group. Conclusion These data therefore suggested that HO-1 provide critical protection against LPS-induced cardiocyte injury. The protection seemed to be mediated through down-regulation of the activity of NF-κB.