Effects of T-cadherin on hypoxia/reoxygenation-induced apoptosis of cardiomyocytes from neonatal rats

AIM: To investigate the mechanism of cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) by silencing a new adiponectin receptor T-cadherin through adenovirus-mediated RNA interference. METHODS: The primary cardiomyocytes were isolated from neonatal rats and cultured for 72 h. The cardiomyocytes were randomly divided into control group, H/R group, APN+H/R group, Ad-T-cadherin-siRNA+APN+H/R group and Ad-HK (adenovirus negative control)+APN+H/R group. The transfection ability and efficiency were examined. The expression of T-cadherin at mRNA and protein levels was detected by RT-PCR and Western blotting. The apoptotic rate was analyzed by flow cytometry and TUNEL. RESULTS: High purity of neonatal rat cardiomyocytes was obtained by primary culture. After 48 h, over 90% of myocardiocytes were infected at MOI=100. The transfected myocardiocytes showed a low expression level of T-cadherin under normal physiological condition. Compared with APN+H/R group, the cell apoptotic rate significantly increased in Ad-T-cadherin-siRNA+APN+H/R group (P<0.05). Compared with H/R group, the difference was not statistically significant (P>0.05). CONCLUSION: Ad-T-cadherin-siRNA effectively infects myocardial cells in vitro and successfully reduces the expression of T-cadherin in myocardial cells. The inhibitory effect of adiponectin on H/R-induced cardiomyocyte apoptosis is attenuated by decreasing the expression of T-cadherin.     

Establishment and application of cell model for P2ry2 modulators screening based on CaCC

AIM To construct a high-throughput screening cell model for P2Y₂ purinergic receptor （P2ry2） modulators based on calcium-activated chloride channel （CaCC）. METHODS The mRNA expression of P2ry2 in Fischer rat thyroid （FRT） cells was detected by RT-PCR， and the PCR products were collected and sequenced. The protein expression of P2ry2 in the FRT cells was also detected by Western blot. The eukaryotic expression vectors ANO1 and YFP-H148Q/I152L were constructed. The FRT cells co-expressing ANO1 and YFP-H148Q/I152L were obtained by liposome transfection， antibiotic screening and limited dilution. The expression of ANO1 and YFP-H148Q / I152L in the cells was observed under fluorescent inverted microscope. The validation of the cell model for screening P2ry2 modulators was verified by the fluorescence quenching kinetics tests， and intracellular free calcium was analyzed by Fura-2 staining to investigate the dose-dependent relationship between intracellular calcium concentration and P2ry2 modulators. Z' factor was applied to evaluate the stability and repeatability of the cell model. RESULTS P2ry2 were endogenously expressed in the FRT cells. The expression of ANO1 on the cell membrane and the expression of YFP-H148Q/I152L in the cytoplasm were observed under fluorescent inverted microscope. The cell model was successfully constructed. The fluorescence quenching kinetics test confirmed the cell model for screening P2ry2 modulators was constructed successfully， and the calcium concentration in cytoplasm was increased rapidly after the addition of a small amount of P2ry2 agonist， indicating that the cell model was sensitive for detecting the calcium concentration in cytoplasm. The calcium concentration in cytoplasm， P2ry2 modulators and the slope of fluorescence change were in a dose-dependent manner， respectively. The Z' factor was 0.75， indicating that the established cell model was able to use for high-throughput screening of P2ry2 modulators with excellent stability and repeatability. CONCLUSION A simple， economical， and efficient cell screening model of P2ry2 modulators is successfully constructed， which is suitable for the detection and evaluation of P2ry2 modulators.     

Effects of myofibrillogenesis regulator on myocardial hypertrophy

AIM: To investigate the effects of myofibrillogenesis regulator-1 (MR1) on myocardial hypertrophy. METHODS: Three stem-loop structures of rMR1 mRNA were selected as targets to establish RNA interference carriers. After transient transfection with plasmids, cultured cardiomyocytes of neonatal were used to perform RT-PCR for choosing the first target to carry out RNA interference blocking MR1 gene. In order to observe the effect of MR1 gene silence on myocardial hypertrophy induced by angiotensin Ⅱ (AngⅡ), the radiation intensity of tritium-leucine (［3H］-Leu) was used to label the cardiomyocytes. Morphological observation, protein extraction and Western blotting were also used to investigate protein synthesis rate, cell surface area and expression of rMR1. RESULTS: The radiation intensity of tritium-Leucine in AngⅡ group increased 21.4% (P<0.01), the cell surface area increased 65.8% (P<0.01) and the expression of rMR1 was up-regulated. Captopril, an inhibitor of angiotensin converting enzyme, abolished the hypertrophy effect and expression of rMR1 induced by AngⅡ. After MR1 gene blocking, the radiation intensity and cell area decreased 30.2% and 31.1% (P<0.01), respectively, compared to AngⅡ group. The expression of rMR1 was depressed. CONCLUSIONS: AngⅡ induces myocardial hypertrophy and upregulates expression of rMR1. Preconditioning with captopril eliminates the effect of AngⅡ, indicating that the increased expression of rMR1 is correlated with myocardial hypertrophy induced by AngⅡ. Blocking of MR1 gene abolishes the effect of AngⅡ and depresseses the expression of rMR1, the effect is similar to ACEI, indicating that MR1 takes part in the procession of hypertrophy through promoting the synthesis of contractive protein in cardiomyocytes.     

PERK-mediated endoplasmic reticulum stress is involved in angiotensin II-induced myocardial hypertrophy

AIM: To investigate the role of protein kinase R-like endoplasmic reticulum kinase (PERK)-mediated endoplasmic reticulum stress (ERS) in angiotensin II (AngⅡ) -induced myocardial hypertrophy. METHODS: In the hypertrophy model of AngⅡ-induced cardiomyocytes isolated from neonatal Sprague-Dawley rats, the methods of morphological observation, [³H]-leucine incorporation and surface area measurement were employed to assess the cardiomyocyte hypertrophy. Real-time PCR, RT-PCR and Western blotting were used to detected the expression of glucose-regulated protein 78 (GRP78), calreticulin (CRT), PERK, eukaryotic initiation factor 2α (eIF2α) and C/EBP homologous protein (CHOP) at mRNA and protein levels. RESULTS: Compared with control group, Ang II-treated cardiomyocytes showed that the mRNA and protein expression of CRT increased by 146.4% and 125.3%, respectively (P<0.05). The mRNA and protein expression of GRP78 increased by 84.0% and 77.6%, respectively (P<0.05). The mRNA and protein expression of PERK increased by 165.4% and 132.1%, respectively (P<0.05).The mRNA and protein expression of eIF2α was increased by 110.9% and 46.5%, respectively (P<0.05). The mRNA and protein expression of CHOP also increased by 117.7% and 63.3%, respectively (P<0.05). CONCLUSION: PERK-mediated ERS response is involved in AngⅡ-induced cardiomyocyte hypertrophy.     

Construction of Pax-8 gene recombinant plasmid and study of its function

AIM: To investigate the effects of over-expression of Pax-8 gene on the proliferation and apoptosis of H9c2 cells(a cardiomyocyte cell line). METHODS: The full length of rat Pax-8 gene was restrictively digested by Kpn I and Not I from the pCMV sport6-Pax-8 vector, and then inserted into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)-Pax-8 was confirmed by restriction endonuclease digestion and sequencing. The pcDNA3.1(+)-Pax-8 was transfected into H9c2 cells. The expression of Pax-8 at mRNA and protein levels was identified after transfection by RT-PCR and Western blotting. The cell proliferation was measured by CCK-8. Cell apoptosis was induced by serum deprivation in H9c2 cells transfected with Pax-8 gene. The apoptosis rate of the cells was determined by flow cytometry with annexin V-FITC and propidium iodide double staining. The protein expression of activated caspase-3 was measured by Western blotting. RESULTS: The full length of Pax-8 gene was successfully cloned into pcDNA3.1(+) expression vector and over-expression of Pax-8 at mRNA and protein levels was observed in H9c2 cells transfected with Pax-8 gene as compared to the wild-type cells and the cells transfected with an empty vector (both P<0.05). Transfection of Pax-8 gene promoted the proliferation of the cardiomyocytes (P<0.05) and inhibited the apoptosis rates induced by serum deprivation (P<0.01). The expression level of activated caspase-3 was increased by serum deprivation and attenuated by Pax-8 transfection (P<0.01). CONCLUSION: The pcDNA3.1(+)-Pax-8 expression vector was successfully constructed and over-expression of Pax-8 gene in cardiomyocytes is obtained. Pax-8 gene acts as an anti-apoptotic factor in cardiomyocytes by promoting cell proliferation and inhibiting apoptosis.     

Cardiomyocyte apoptosis and expression of Bcl-2, Bax protein after acute myocardial infarction and late reperfusion in dogs

AIM: To study the effect of late reperfusion on apoptotic cardiomyocytes in the risk area of acute myocardial infarctin in dogs. METHODS: The experiment was divided into three groups: sham operation group, acute myocardial infarction (AMI) group, and late reperfusion (LR) group. Apart from sham operation group, the other two groups were subjected to left anterior descending branch of coronary artery ligation. The acute myocardial infarction group was only subjected to ligation for 12 hours, late reperfusion group was subjected to ligation for 6 hours following by 6 hours of reperfusion. The cardiomyocyte apoptosis was measured by TUNEL assay. Immunohistochemistry and Western blotting analysis were used to detect the expression of Bcl-2 and Bax protein. RESULTS: The number of apoptotic cardiomyocytes in late reperfusion group was much less than acute myocardial infarction group (P<0.05), and increased significantily as compared with sham operation group (P<0.01). The expression of Bcl-2 protein was enhanced gently in late reperfusion group in contrast to acute myocardial infarction group, but no significant difference in the two groups (P>0.05) was observed, although it was much more in the two groups than that in sham operation group (P<0.01). The expression of Bax protein in late reperfusion group was much higher than that in sham operation group (P<0.01), and was lower than that in acute myocardial infarction group (P<0.05). CONCLUSION: Late reperfusion reduces cardiomyocyte apoptosis in the risk area of acute myocardial infarction. The mechanism may be that late reperfusion can decrease the expression of Bax protein.     

Fuzi polysaccharide protects neonatal rat cardiomyocytes with hypoxia-reoxygenation by inhibiting endoplasmic reticulum stress

AIM: To investigate whether the protection mechanism of Fuzi polysaccharide (FPS) is related to inhibition of endoplasmic reticulum stress in cultured neonatal rat cardiomyocytes with hypoxia/reoxygenation (H/R). METHODS: Cultured rat myocardial cells were divided into control group, H/R group (hypoxia for 3 h and reoxygenation for 6 h) and different concentrations of FPS (0.1 g/L, 1 g/L, 10 g/L or 20 g/L) +H/R groups. The cell survival was detected by MTT assay and cell apoptosis of cardiomyocytes was measured by flow cytometry using Annexin V-FITC staining. The expression of glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12 were determined by Western blotting. The mRNA expression of CHOP and caspase-12 was detected by quantitative PCR. RESULTS: After reoxygenation, the expression of GRP78, CHOP and caspase-12 in cardiomyocytes was increased. Compared with H/R group, the expression of GRP78, CHOP and caspase-12 in FPS+H/R groups was significantly inhibited, the survival rate of cardiomyocytes was increased and the apoptosis of cardiomyocytes was inhibited. This protective effect of FPS was in a dose-dependent manner and reached its peak at 10 g/L. CONCLUSION: Fuzi polysaccharide protects cardiomyocytes from H/R injury. The mechanism is related to inhibiting endoplasmic reticulum stress.     

Effects of adiponectin on reducing endoplasmic reticulum stress injury induced by hypoxia-reoxygenation in cultured rat neonatal cardiomyocytes

AIM: To investigate the effects of adiponectin (APN) on hypoxia-reoxygenation (H/R) induced endoplasmic reticulum stress injury in cultured cardiomyocytes. METHODS: Primary cultured cardiomyocytes were obtained from neonatal rats by enzymatic digestion method. The α-actin expression as molecular marker of the cardiomyocytes was observed by immunocytochemistry. The cells cultured for 72 h were used in the experiment and divided into groups randomly: control group, H/R group, APN+H/R (3 mg/L, 10 mg/L, 20 mg/L, 30 mg/L) groups. The morphological changes of the cardiomyocytes were observed under phase contracted microscope. The content of LDH was measured. The cardiomycocyte apoptosis was detected by flow cytometry. The expression levels of GRP78 and caspase-12 were examined by RT-PCR and Western blotting. RESULTS: The apoptotic rate was significantly increased in H/R group as compared to that in control group (68.20%±1.73% vs 0.73%±0.21%, P<0.05). The levels of LDH in H/R group were also significantly increased. Compared to untreated cells, the protein and mRNA levels of GRP78 and caspase-12 increased significantly in H/R cells. The APN preconditioning significantly reversed these changes. The indexes above improved obviously as compared to H/R group (P<0.05) in a dose-dependent manner. CONCLUSION: Hypoxia/reperfusion induces endoplasmic reticulum stress in rat cardiomyocytes. Adiponectin decreases the endoplasmic reticulum stress injury and plays a protective role by extenuation of cadiomyocyte apoptosis.     

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.     

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.     

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.     

Anti-cardiac myosin heavy chain antibodies isolated from patients with acute myocardial infarction induce rat cardiomyocyte apoptosis

AIM: To study the effect of human anti-cardiac myosin heavy chain antibodies (AMHCA) on rat cardiomyocyte apoptosis. METHODS: Rat cardiomyocytes were isolated by the method of enzymolysis. Apoptosis of the cardiomyocytes was observed and measured by DNA end labelling and Annexin-V/PI double-staining assay. The proteins levels of apoptosis related P53 and Bcl-2 and the second messenger calcium were measured by Western blotting, patch clamp and confocal calcium imaging, respectively. RESULTS: AMHCA was able to induce cardiomyocyte apoptosis in a dose dependent manner. In the presence of AMHCA, apoptosis-accelerating nucleoprotein P53 promoted myocardial apoptosis, while apoptosis-inhibiting cytoplasmic protein Bcl-2 inhibited myocardial apoptosis. Meanwhile, the concentration of cytoplasmic calcium was elevated. No effect of AMHCA on L-type calcium currents was observed. CONCLUSION: As a novel triggering factor, AMHCA isolated from the patients with AMI induces cardiomyocyte apoptosis.     

Effects of aplastic anemia patient serum on the expression of the crucial cyclin D isoform in cord blood CD34+ cells

AIM:To explore the pathogenesis of aplastic anemia (AA), we identified the crucial isoform of cyclin D that determine the proliferation of the cord blood CD34+ cells and observed effects of AA serum on the expression of crucial cyclin D isoform in umbilical cord blood CD34+ cells. METHODS:The CD34+ cells were isolated with MIDI-MACS system. The isoforms of cyclin D were detected by RT-PCR and Western blotting. Methylcellulose culture system was used to measure the formation of CFU-GM. The expression level of crucial cyclin D isoform was assayed by RT-PCR and Western blotting after the CD34+ cells were incubated in AA serum. RESULTS:The crucial cyclin D isoform in CD34+ cells was cyclin D3. The AA serum inhibited the formation of CFU-GM and down-regulated expression level of the cyclin D3 from the mRNA to protein level, respectively. CONCLUSION:The AA serum inhibits the proliferation of CD34+ cells and down-regulates level of cyclin D3, this may be one of hematopoiesis inhibition mechanisms in AA.     

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.     

Gene expression of spleen lymphocytes induced by gp96-peptide complexes and its anti-tumor effect in vitro

AIM: To study the expressing variation of TNF-α and IFN-γ mRNA in mouse splenocytes induced by H22 tumor cells derived heat shock protein gp96-peptide complexes in vitro,and to observe the morphologic change of H22 tumor cells which treated with the culture supernatant.METHODS: H22 tumor cells derived HSP gp96 was obtained by the techniques for protein extraction and purification and was identified by Western blotting method.The expression values of TNF-α and IFN-γ mRNA in spleen lymphocytes were detected by semi-quantitative RT-PCR.The morphologic changes of H22 tumor cells induced by the culture supernatant were observed by laser scanning confocal microscopy (LSCM) and transmission electron microscope (TEM).RESULTS: Purified heat shock protein gp96 was identified by Western blotting.The expression value of TNF-α and IFN-γ mRNA in activated spleen lymphocytes induced by gp96-peptide complexes was higher than that in control groups (P<0.05).The morphologic change of apoptosis of H22 tumor cells,which treated by the culture supernatant of experimental group was observed with LSCM and TEM.CONCLUSION: Heat shock protein gp96-peptide complexes increase the expression value of TNF-α and IFN-γ mRNA in spleen lymphocytes of mouse in vitro.Besides,apoptosis of H22 cells is induced by immunologic active material secreted by activated splenocytes.     

Expression of aquaporin 3 in human prostate

AIM: To determine the expression of aquaporin 3 (AQP3) in transitional and peripheral zone of human prostate tissue.METHODS: AQP3 mRNA expression was analyzed in five prostate tissues by RT-PCR. AQP3 protein expression and localization were characterized by Western blotting and immuno-staining with polyclonal anti-AQP3 antibody.RESULTS: The results of RT-PCR and Western blotting showed that AQP3 was expressed in transitional and peripheral zone of prostate tissue. The AQP3 protein expression in transitional zone was higher than that in peripheral zone. The results of immuno-staining indicated that AQP3 protein was expressed in secretory cells of prostate.CONCLUSION: The AQP3 was expressed in prostate tissue, suggesting that AQP3 play an important role in the secretion of prostate liquid.