Effects of beta2-adrenergic blocker on cytosolic Ca2+ in isolated cardiomyocytes of rats with myocardial infarction

AIM: To investigate if beta2-adrenergic receptors result in more Ca2+ load after myocardial infarction (MI), the effects of beta2-adrenergic blocker on cytosolic Ca2+ (［Ca2+］i) were studied. METHODS: Male Wistar rats underwent a ligation of left coronary artery (n=9) or a sham operation (n=3). Cardiomyocytes were dissociated at 2, 4 and 8 weeks after MI and ［Ca2+］i was measured via fura-2 fluorescence. The response of cardiomyocytes to isoproterenol (1 μmol/L) in the presence or absence of atenolol (1 μmol/L), beta2-adrenergic blocker ICI118,551 (0.1 μmol/L) or propranolol (1 μmol/L) was examined. RESULTS: ICI118,551 suppressed the increase in ［Ca2+］i induced by isoproterenol at 4 and 8 weeks after MI (24.5%±5.7% vs 57.8%±13.2%, P<0.01; 12.2%±7.9% vs 44.6%±11.3%, P<0.01), but had no effects in control and 2 weeks post-MI groups. It decreased ［Ca2+］i in control and the three post-MI groups by 14.3%, 7.9%, 57.6% and 72.6%, respectively. Atenolol had suppressive effects only in control and 2 weeks post-MI groups (P<0.05). Propranolol had suppressive effects in control and all three post-MI groups (P<0.01). CONCLUSION: Beta 2-adrenergic blocker ICI118,551 exerts negative effects on ［Ca2+］i after MI, and the effects dramatically increase with the progression of MI.     

Bio-effects of salusins on isolated rat heart and neonatal cardiomyocytes

AIM: To investigate the bio-effects of salusins on rat heart and cardiomyocytes. METHODS: The cardiac function was determined by multipurpose polygraph in isolated rat heart treated with various concentrations of salusin-α or salusin-β.[⁴⁵Ca²⁺] and[³H]-Leu incorporation were determined in cultured neonatal rat cardiomyocytes with β-liquid scintillation counter. RESULTS: 10^-12-10^-7mol/L salusin-α and salusin-β had no effects on isolated rat cardiac function. However, salusin-α and salusin-β stimulated uptake and[³H]-Leu incorporation. The [⁴⁵Ca²⁺] uptake induced by salusins were inhibited by nicardipine, and were synergistically increased by endothelin-1. The[³H]-Leu incorporation induced by salusin-α and salusin-β was inhibited by nicardipine, FK506 (a special inhibitor of carcineulin), PD₉₈₀₅₉ (inhibitor of MAPK) and chelerthine (inhibitor of PKC). The effects of salusin-β[⁴⁵Ca²⁺] on uptake was stronger than those of salusin-α. But there were no statistical difference in[³H]-Leu incorporation between salusin-α and salusin-β. CONCLUSIONS: Salusin-α and salusin-β did not affect directly cardiac function in rat hearts. But salusins improved calcium uptake and protein synthesize in neonatal rat cardiomyocytes. Those effects of salusins were related with calcium channel, carcinuelin, MAPK and PKC signal pathways. Salusins may be the regulatory factors for myocardium growth and hypertrophy.     

Inhibitory effect of hypoxia preconditioning on hypoxia/reoxygenation-induced apoptosis in cardiomyocytes

AIM: To study the role of hypoxia preconditioning (HP) in hypoxia-reoxygenation (HR)-induced apoptosis in neonatal rat cardiomyocytes and the possible mechanisms. METHODS: Cultured neonatal rat cardiomyocytes were divided into three groups: normal group, HP+H/R group and H/R group. Acridine orange (AO) staining was performed to detect morphological changes of apoptotic cells. Apoptosis rates of cardiomyocytes were detected by flow cytometry. Colorimetric assay was used to detect caspase-3 activity. Expression of Bcl-2 protein was detected by immunohistochemistry combined with computer image analysis. RESULTS: Apoptotic cells were detected by AO staining after hypoxia of 6 h followed by 3 h-reoxygenation. The hypodiploid apoptotic peak was detected by flow cytometry with the apoptotic rates of (29.7±5.4)%. A significantly reduced apoptotic rates of (7.8±1.3)% was detected in HP group（P＜0.01）. The caspase-3 relative activity of cardiomyocytes induced by H/R was 5.9±0.8, significantly higher than that of control group. HP markedly reduced caspase-3 relative activity to 2.6±0.5 in contrast with H/R group （P＜0.01）. Bcl-2 protein was positive in normal cardiomyocytes with an A value of 119.4±7.1. The A value of H/R group was 99.6±5.0, significantly lower than that in normal group （P＜0.01）. The A value of HP+H/R group was 126.5±6.2, significantly higher than that in H/R group（P＜0.01）. CONCLUSION: HP inhibits H/R-induced apoptosis of cardiomyocytes by improving the expression of Bcl-2 and reducing caspase-3 activity.     

Cyclosporine A suppresses the hypertrophic effect of neuropeptide Y in rat cardiomyocytes

AIM:To investigate the effect of cyclosporine A on rat cardiomyocyte hypertrophy caused by neuropeptide Y (NPY).METHODS:Cardiomyocytes of neonatal Wistar rats were cultured with NPY or NPY together with CsA. For assessing protein synthesis rate and c-jun mRNA expression in cardiomyocytes, the methods of [³H]-Leu incorporation and RT-PCR were used.RESULTS:(1) [³H]-Leu incorporation in cardiomyocytes: [³H]-Leu incorporation in NPY (10 nmol/L) group was higher than that in control group, but there were no distinct changes between two groups. To compare with control group, [³H]-Leu incorporation in NPY (100 nmol/L) group were increased significantly (P＜0.05). There was no significant change between control group and CsA group; (2) c-jun mRNA expression in cardiomyocytes: RT-PCR production of c-jun mRNA in NPY group was enhanced considerably compared with CsA group and control group (P＜0.01). There was no significant change between CsA group and control group.CONCLUSIONS:NPY can induce cardiomyocyte hypertrophy. Cyclosporine A (inhibitor of CaN) can blunt the effect of NPY, suggesting that the Ca²⁺/CaM-dependent calcineurin (CaN) signaling pathway plays an important role in it.     

Inhibitory effect of iron on vasodilatation in the isolated rat aorta

AIM: The objectives of the present study were to examine the effect of iron on relaxation of isolated rat aortic rings, and to elucidate the underlying mechanism. METHODS: The thoracic aortic rings of male Sprague-Dawley rats were mounted on bath system. Vasodilatation of aortic rings preconstricted with 10^-6 mol/L of phenylephrine (PE) was measured. RESULTS: (1) Exposure of endothelium-intact aortic rings to ferric ammonium citrate (FAC) for 30 min caused a significant reduction in the relaxation response to acetylcholine (ACh). Pretreatment with L-arginine (L-Arg) before incubation with FAC did not reverse the inhibition of relaxation response to ACh completely. (2) In endothelium-intact aortic rings, L-Arg relaxed the PE preconstricted vessels. Exposure to FAC for 30 min caused the decrease in the relaxation response to L-Arg. There was no difference in the relaxation response to nitric oxide donor, sodium nitroprusside, between endothelium-denuded arteries treated with or without FAC. (3) Dimethyl sulfoxide had no effect on the inhibition of relaxation to ACh by FAC in endothelium-intact rings. Pretreatment of arteries with glutathione and catalase prevented the decrease in relaxation responses to ACh induced by FAC. (4) The nitric oxide synthase activity was (56.49±2.49)×10³U/g protein in normal aorta with endothelium, while after incubation with FAC for 30 min, it reduced to (25.15±5.75)×10³U/g protein ( P< 0.05). CONCLUSION: Inactivation of nitric oxide synthase and decrease in intracellular glutathione level might mediate iron-induced inhibition of arterial relaxation responses to ACh.     

Dendranthema morifolium attenuated the reduction of contraction of isolated heart and cardiomyocytes induced by ischemia/reperfusion

AIM: To investigate the effect of Dendranthema morifolium (Ramat) Tzvel (DM) on isolated rat heart and ventricular myocytes during ischemia/anoxia and reperfusion/reoxygenation.METHODS: The Langendorff perfused rat hearts were used to measure intraventricular pressure and coronary flow. The cell contraction and intracellular calcium transient in enzymatically isolated ventricular myocytes were determined. RESULTS: (1) DM (0.5 g/L) significantly attenuated the inhibitory effects induced by ischemia/reperfusion on left ventricular developed pressure (LVDP), ±dp/dt_max, coronary flow and LVDP×HR, meanwhile increased the content of SOD and decreased the content of MDA in the myocardium; (2) DM (0.5 g/L) attenuated the inhibitory effects of anoxia and reoxygenation on i transient and cell contraction in isolated ventricular myocytes. CONCLUSION: DM attenuated the effects on contractility and intracellular calcium induced by ischemia/anoxia and reperfusion/reoxygenation in the isolated rat heart and the ventricular myocytes. The mechanism might be related to increase in SOD activity and maintaining [Ca]i homeostasis.     

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.     

Effect of carvedilol on store overload-induced Ca2+ release in rat pacing cardiomyocytes

AIM：To explore the effect of carvedilol on store overload-induced Ca2+ release (SOICR) in pacing cardiomyocytes. METHODS：Single rat cardiomyocyte with rapid pacing was perfused with isoprenaline and caffeine to induce calcium overload. Spontaneous calcium releases through sarcoplamic reticulum calcium release channel (ryanodine receptor 2, RyR2) were investigated by the method of fluorescence imaging. The cardiomyocytes were divided into control (DMSO) group, carvedilol group, metoprolol group, phentolamine group and nifedipine group. RESULTS：In control group, the incidence of SOICR in cardiomyocytes was significantly increased under the condition of calcium overload by perfusing with isoprenaline and caffeine in addition to the enhancement of calcium transient. The incidence of SOICR in carvedilol group was significantly lower than that in control group at the pacing frequency of 1 Hz to 4 Hz (2.00%, 6.00%, 10.00% and 16.00% vs 43.59%, 74.36%, 87.18% and 89.71%, respectively, P<0.01). The inhibitory effect of carvedilol was not significantly different at variant pacing frequency (P>0.05). The incidences of SOICR in metoprolol group, phentolamine group and nifedipine group had no significant difference compared with control group (P>0.05). The amplitude of calcium transient and caffeine peaking value of pacing cardiomyocytes had no significant difference among different groups (P>0.05). CONCLUSION：Carvedilol effectively suppresses SOICR in pacing cardiomyocytes due to its direct inhibition on the spontaneous opening of the cardiac RyR2 channel rather than the α1, β1 receptor or L-type calcium channel blockade.     

Role of Nrf2-ARE signaling pathway in protective effect of hypoxia or pinacidil postconditioning against hypoxia-reoxygenation injury in adult rat cardiomyocytes

AIM：To investigate whether nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is involved in the protective effect of hypoxia or pinacidil postconditioning against hypoxia-reoxygenation injury in adult rat cardiomyocytes. METHODS：Cardiomyocytes were isolated from the left ventricle of male adult Sprague-Dawley rats (250~300 g) by Langendorff perfusion and collagenase II digestion. The cells were randomly divided into six groups (n=8 in each group). The cells in N group were cultured for 22 h without any treatment. The cells in the other five groups were cultured for 20 h and then underwent 45 min of hypoxia followed by 60 min of reoxygenation (M group), three 5-min cycles of reoxygenation/hypoxia plus 60 min of reoxygenation (IPO group), or treatment with pinacidil (25, 50 and 100 μmol/L) for 5 min plus 60 min of reoxygenation (P25, P50 and P100 groups, respectively). The expression of Nrf2, NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1) at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. RESULTS：The mRNA and protein levels of Nrf2, HO-1, NQO1 and SOD1 in M, IPO, P25, P50 and P100 groups were significantly decreased compared with N group (P＜0.05), and those in IPO, P25, P50 and P100 groups were obviously higher than those in M group (P＜0.05). The mRNA levels of Nrf2, HO-1, NQO1 and SOD1 in IPO and P50 groups were obviously higher than those in P25 and P100 groups (P＜0.05). The mRNA expression of SOD1 in P25 group was obviously higher than that in P100 group (P＜0.05). The protein levels of Nrf2, NQO1 and SOD1 in IPO and P50 groups were obviously higher than those in P25 and P100 groups (P＜0.05). The protein expression of HO-1 in P50 group was obviously higher than that in IPO, P25 and P100 groups (P＜0.05). The protein levels of HO-1 and NQO1 in P25 group were obviously higher than those in P100 group (P<0.05). CONCLUSION：Hypoxia or pinacidil postconditioning may attenuate hypoxia-reoxygenation injury in adult rat cardiomyocytes via activation of Nrf2-ARE signaling pathway.     

Role of aloperine on ischemia-reperfusion-induced injury and inflammatory response in H9c2 cardiomyocytes

AIM: To explore the role of aloperine in ischemia-reperfusion (I/R)-induced H9c2 cardiomyocyte injury and inflammation.METHODS: The H9c2 cardiomyocytes were cultured under hypoxia and re-oxygenation conditions to simulate ischemia-reperfusion (SI/R) injury. After treatment with aloperine at various doses, the cell viability was measured by MTT assay. The cell apoptosis was analyzed by flow cytometry. Simultaneously, the levels of lactate dehydrogenase (LDH), malonaldehyde (MDA) and caspase-3 activity were detected by the commercial kits. The levels of inflammatory cytokines were also detected by ELISA. Moreover, the effects of aloperine on the activation of PI3K/AKT signaling pathway were determined by Western blot.RESULTS: Pre-treatment with aloperine remarkably abated the inhibitory effect of SI/R on H9c2 cell viability, and decreased the elevations of LDH and MDA triggered by SI/R (P<0.05). Pre-treatment with aloperine dramatically suppressed the cell apoptosis induced by SI/R treatment (P<0.05), concomitant with the decrease in caspase-3 activity and increase in Bcl-2/Bax ratio (P<0.05). In contrast to SI/R group, aloperine treatment notably restrained the concentrations of pro-inflammatory cytokines, including interleukin-6, tumor necrosis factor-α and interleukin-1β (P<0.05). Furthermore, aloperine remarkably increased the protein levels of p-PI3K and p-AKT. While blocking the PI3K/AKT pathway with its specific inhibitor LY294002, the viability-promoting, anti-apoptotic and anti-inflammatory effects of aloperine on the H9c2 cells were obviously attenuated (P<0.05).CONCLUSION: Alope-rine protects against cardiomyocytes from I/R injury and inhibits inflammatory responses by activating the PI3K/AKT signaling pathway, implying a potential benefic role of aloperine against myocardial I/R injury.     

番木瓜内生细菌MG-Y2的鉴定及其生防作用

为筛选具有防病作用的植物内生菌,采用组织分离法和稀释分离法,分离番木瓜果皮中的内生细菌,得到103个菌株。采用培养基平板抑菌圈测定法从这些菌株中筛选到1株具有拮抗活性的细菌MG-Y2,对番木瓜疫霉病菌(Phytophthora nicotianae)、番木瓜炭疽病菌(Colletotrichum gloeosporioides)等8种病原菌有较强的拮抗作用。通过对其形态特征和生理生化特性测定以及16SrDNA部分序列同源性分析,鉴定该细菌为恶臭假单胞菌生物变种Ⅰ(Psudomonas putida biovarⅠ)。采用喷雾接种处理,MG-Y2可进入番木瓜叶片、叶柄、果皮和果肉中定殖。进行番木瓜果实采后防病试验,MG-Y2对采后番木瓜疫病和炭疽病的防治效果分别达到88.8%和57.4%。试验结果显示MG-Y2具有潜在的生防应用价值。     

Effects of norepinephrine on the activity of NF-κB in cardiomyocytes

AIM: To study the effects and mechanisms of norepinephrine (NE) on the activity of nuclear factor-kappa B (NF-κB) in cardiomyocytes. METHODS: Using the cultured neonatal rat cardiac myocytes, the levels of reactive oxygen species and the nuclear DNA binding activity of NF-κB were measured in cardiomyocytes before and after stimulated with NE alone, NE+prazosin+propranolol, and NE+vitamin E, respectively. RESULTS: NE increased significantly the levels of reactive oxygen species and NF-κB activity in cardiac myocytes. These effects of NE were attenuated by vitamin E pretreatment, as well as prazosin and propranoloe. CONCLUSION: The effects of NE on cardiomyocytes might be exerted partly through NF-κB activation, associated with NE-induced overproducion of reactive oxygen species via the adrenergic receptor pathway.     

Effects of cycloastragenol on cardiac fibrosis induced by isoproterenol in mice

AIM: To investigate the effects of cycloastragenol (CAG) on cardiac fibrosis in mice and the mechanism involved. METHODS: The mouse model of cardiac fibrosis induced by isoproterenol (ISO) was established and treated with high- and low-dose CAG. Cardiac function was measured by echocardiography, and heart sections were stained by Masson’s trichrome for fibrosis assessment. The expression of fibrosis-related factors was assayed using RT-qPCR, Western blot and immunohistochemistry. In addition, cardiac fibroblasts isolated from neonatal factors rat ventricles were cultured and administrated with ISO followed by CAG treatment, and then the expression profile of the factors above was assayed using RT-qPCR. RESULTS: Treatment with CAG significantly alleviated ISO-induced cardiac dysfunction and fibrosis, inhibited the mRNA expression of oxidative stress-related factors NADPH oxidase 4 and inducible nitric oxide synthase, and blocked the phosphorylation of proteins associated with nuclear factor-κB signaling pathway as well as the expression of transforming growth factor-β (TGF-β). In addition, it was demonstrated that CAG also inhibited the mRNA expression of the factors above in primary cardiac fibroblasts administrated with ISO. CONCLUSION: CAG markedly rescues ISO-induced cardiac dysfunction and fibrosis via inhibition of oxidative stress, inflammation and TGF-β levels.     

Relaxation effect of isoliensinine on isolated mouse airway smooth muscle

AIM: To study the relaxation effect of isoliensinine on high K⁺-induced isolated mouse airway smooth muscle (ASM) and the underlying mechanism. METHODS: The muscle tension transducer was used to detect the effects of isoliensinine on high K⁺-induced precontraction and Ca²⁺ influx in ASM. The technique of patch-clamp and calcium imaging system were respectively used to examine the effects of isoliensinine on LVDCC currents and[Ca²⁺]_i of the ASM cells (ASMCs). RESULTS: Isoliensinine significantly relaxed precontracted ASM induced by high K⁺ in a concentration-dependent manner. The maximum relaxation ratio was(95.3±3.9)% by isoliensinine at 100 μmol/L. In addition, LVDCC currents were measured using the whole-cell patch-clamp technique, which were abolished by isoliensinine. High K⁺-induced 340/380 nm fluorescence ratio of Fura-2 was 0.63±0.10 in ASMCs, while it decreased to 0.36±0.05 after the addition of isoliensinine (P<0.01). When isoliensinine was added at the peak point of[Ca²⁺]_i, the ratio rapidly decreased from 0.74±0.02 to 0.42±0.05 (P<0.01). Moreover, isoliensinine inhibited high K⁺-induced Ca²⁺ influx-mediated contraction of ASM. CONCLUSION: Isoliensinine inhibits LVDCC currents, terminates Ca²⁺ influx and reduces[Ca²⁺]_i, eventually resulting in relaxation of the ASM, indicating isoliensinine might be a potential bronchodilator.     

Effect of polyamine on cardiac hypertrophy induced by isoproterenol in rats

AIM: To investigate the effect of polyamine, ornithine decarboxylase（ODC）and spermidine/spermine N1-acetyltransferase (SSAT) cardiac hypertrophy induced by isoproterenol (ISO) in rats. METHODS: Cardiac hypertrophy in rats was induced by subcutaneous administration of isoproterenol. High performance liquid chromatography was used to measure the concentrations of polyamines. RT-PCR and Western blotting were used to determine the mRNA and protein expressions of ODC and SSAT during different time points. RESULTS: Compared with control group, heart weight index was increased markedly at ISO 7 d. The concentration of putrescine was increased at ISO 1 d (P<0.05) and increased significantly at 5d and 7 d (P<0.01). The concentration of spermidine was increased at 3 d and increased significantly at 7 d. The concentration of spermine was increased and total pool of polyamine was increased significantly. The expression of ODC and SSAT mRNA were upregulated at 1 d (P<0.05) and kept on high level (P<0.01). The expressions of ODC and SSAT protein were upregulated at 1 d and 5 d (P<0.05) and the changes were significant at 7 d (P<0.01). CONCLUSION: These results indicate that increased polyamine and the upregulated expression of ODC and SSAT in rat cardiac tissue may take part in the process of cardiac hypertrophy induced by isoproterenol.     

Vasodilatatory effect and mechanism of CH2Cl2 extract of flos magnoliae on isolated rat thoracic aorta

AIM: To characterize the vasodilatatory effect of CH₂Cl₂ extract of flos magnoliae (CEF) on isolated rat thoracic aorta and to elucidate its possible mechanism. METHODS: The thoracic aorta was isolated from male Sprague-Dawley (SD) rats and the isometric tension of aortic rings with or without endothelium was measured. RESULTS: CEF (0.1-1 000 mg/L) produced concentration-dependent, endothelium-independent relaxations in phenylephrine (PE)-contracted aortic rings. The maximum relaxation induced by CEF was 78.68%±6.03% in endothelium intact rings and 64.98%±13.90% in endothelium removed rings while the forskolin (1 μmol/L)-induced vasodilation was obtained as 100%. The vasodilatatory effect of CEF was not statistically inhibited by 10 μmol/L glibenclamide (Glib), 3 mmol/L tetraethylammonium (TEA), 100 μmol/L BaCl₂ and 10 μmol/L 1H- -oxadiazole- -quinoxalin-1-one (ODQ) in the preparations without endothelium. The CEF pre-treatment significantly inhibited vasoconstrictions to angiotensin Ⅱ (AngⅡ), prostaglandin F_2α, (PGF_2α), dopamine (Dopa), vasopressin (Vaso), 5-hydroxytryptamine (5-HT) and PE by 91.31%, 82.11%, 95.32%, 90.53%, 72.22% and 83.63%, respectively (P<0.01). In Ca²⁺-free medium treated endothelium removed aortic ring, incubation with CEF at concentration of 82 mg/L significantly attenuated intracellular Ca²⁺ release by PE. In Ca²⁺-free + high potassium medium incubated aortic rings without endothelium, CEF (82 mg/L) markedly inhibited potassium-stimulated Ca²⁺-dependent contraction which was mainly due to Ca²⁺ influx (P<0.01).CONCLUSION: CEF induced vasorelaxation is mainly related to interfering intracellular calcium homeostasis by blocking Ca²⁺ influx and intracellular Ca²⁺ release.     

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.     

Roles of microRNA-29a in expression of Bcl-2 and Mcl-1 in rat cardiomyocytes

AIM: To investigate the regulatory mechanisms of microRNA-29a (miR-29a) on the expression of Bcl-2 and Mcl-1 in rat cardiomyocytes (CM cells). METHODS: The CM cells were isolated from the hearts of newborn rats and transfected with miR-29a mimic (100 nmol/L) by Lipofectamine RNAiMAX. The expression of Bcl-2 and Mcl-1 at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and Western blotting. The luciferase assay was performed in HEK293T cells and CM cells, which were co-transfected with plasmid DNA and miRNA using Lipofectamine 2000. RESULTS: Transfection of miR-29a mimics significantly reduced the expression levels of Bcl-2 and Mcl-1 in CM cells as compared with the control cells (P<0.05). In addition, HEK293T cells co-transfected with miR-29a mimic and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid significantly reduced the luciferase activity as compared with control group (P<0.05). While CM cells transfected with miR-29a inhibitor and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid in succession, the luciferase activity was increased inversely (P<0.05). CONCLUSION: miR-29a may regulate apoptosis by targeting the bcl-2 and mcl-1 genes.