L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na/HCO3 cotransport

The sarcolemmal Na⁺/HCO₃⁻ cotransporter (NBC) plays an important role in intracellular pH (pH_i) regulation in the heart. In the present work we studied, in isolated cat ventricular myocytes, the role of Angiotensin II (Ang II) and reactive oxygen species (ROS) production as potential activators of the NBC. pH_i was measured in single cells in a medium with HCO₃⁻ using the fluorescent pH indicator BCECF. The NH₄⁺ pulse method was used to induce an intracellular acid load and the acid efflux (J_H) in the presence of the Na⁺/H⁺ exchanger blocker HOE642 (10 μM) was calculated as indicator of NBC activity. The following J_H data are presented at pH_i of 6.8 ( and ^# indicate p < 0.05 after ANOVA vs. control and Ang II, respectively). The basal J_H (1.03 ± 0.12 mM/min, n = 11) was significantly increased in the presence of 100 nM Ang II (1.70 ± 0.15 mM/min, n = 8). This effect of Ang II was abolished when we added to the extracellular solution 2 mM MPG (ROS scavenger; 0.80 ± 0.08 mM/min, n = 11^#), 300 μM apocynin (NADPH oxidase blocker; 0.80 ± 0.13 mM/min, n = 6^#), 500 μM 5-hydroxidecanoate (mitochondrial ATP dependent K⁺ channel, mK_ATP, blocker; 0.97 ± 0.21 mM/min, n = 9^#), or the inhibitor of the MAP kinase ERK pathway U0126 (10 μM; 0.56 ± 0.18 mM/min, n = 6^#). We also determined the phosphorylation of ERK during the first min of acidosis and we detected that Ang II significantly enhanced the ERK phosphorylation levels, an effect that was cancelled by scavenging ROS with MPG. In conclusion, we propose that Ang II enhances the production of ROS through the activation of the NADPH oxidase, which in turn triggers mK_ATP opening and mitochondrial ROS production (“ROS-induced ROS-release mechanism”). Finally, these mitochondrial ROS stimulate the ERK pathway, leading to the activation of the NBC.     

食管鳞癌中微小核糖核酸-21的作用及对靶蛋白的影响

目的 探讨微小RNA(miRNA)-21对食管鳞癌细胞Eca109和哈萨克族食管癌的促增殖作用及对程序性细胞死亡基因4(PDCD4)表达的影响。方法将食管鳞癌细胞系Eca109分为miRNA-21 Mimics组（转染序列5′-UCAACAUCAGUCUGAUAAGCUA-3′）、miRNA-21抑制剂组（转染序列5′-UAGCUUAUCAGACUGAUGUUGA-3′）、阴性对照组（转染随机序列）和正常对照组（不予转染等任何处理）。细胞按4.5×105个/孔接种于6孔板,基于RNA干扰(RNAi)技术、使用脂质体2000试剂进行细胞转染。采用细胞计数法检测转染后各组细胞的增殖情况。使用实时荧光定量聚合酶链反应(qRT-PCR)检测各组细胞的miRNA-21转录水平。采用Western印迹技术检测转染后各组PDCD4蛋白表达情况。检测18对哈萨克族食管癌及其对应的癌旁正常食管组织中miRNA-21转录和PDCD4蛋白表达情况。结果 转染48 h后,与正常对照组比较,miRNA-21Mimics组Eca109细胞数增加36％(P=0.002),miRNA-21抑制剂组细胞数减少28％(P=0.002),阴性对照组细胞数变化不明显(P=0.515)。转染48 h后,miRNA-21抑制剂组、miRNA-21 Mimics 组、阴性对照组、正常对照组miRNA-21的相对表达量分别为0.37±0.10、9.17±1.08、0.74±0.23和1.04±0.34,PDCD4蛋白相对表达量分别为1.47±0.11、0.61±0.09、0.89±0.12、0.79±0.02。与正常对照组比较,miRNA-21抑制剂组miRNA-21相对表达量下降(P=0.031)、PDCD4蛋白相对表达量上调（P=0.001）,miRNA-21 Mimics组miRNA-21相对表达量上升(P=0.001)、PDCD4相对表达量下调(P=0.030),阴性对照组则差异无统计学意义（P值分别=0.272和0.541）。18对哈萨克族食管癌组织中16对的miRNA-21相对表达量(0.11±0.09)高于其对应的癌旁正常食管组织(0.03±0.03,P=0.001),PDCD4蛋白相对表达量（0.92±0.39）低于其对应的癌旁正常食管组织（1.57±0.80,P=0.004）,且癌组织中miRNA-21相对表达水平越高,PDCD4蛋白相对表达水平越低（r=-0.538,P=0.046）。结论miRNA-21可能通过抑制PDCD4蛋白表达而促进食管鳞癌细胞Eca109增殖,并且参与哈萨克族食管癌的发生。     

Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.     

ENST00000418539.1调控miR-24对H2O2诱导心肌细胞氧化应激损伤的影响

目的探讨长链非编码RNA ENST00000418539.1(LncRNA ENST00000418539.1)是否通过调控miR-24的表达影响过氧化氢(H₂O₂)诱导心肌细胞氧化应激损伤。方法体外培养大鼠心肌细胞H9c2,200μmol/L H₂O₂处理心肌细胞48 h建立模型,分别将乱序无意义阴性序列(si-NC)、ENST00000418539.1小分子干扰RNA(si-ENST00000418539.1)、si-ENST00000418539.1与阴性对照(anti-miR-NC)、si-ENST00000418539.1与miR-24特异性寡核苷酸抑制剂(anti-miR-24)转染至心肌细胞,使用H₂O₂处理心肌细胞。实时荧光定量聚合酶链反应检测ENST00000418539.1、miR-24的表达量;流式细胞术检测细胞凋亡率;检测丙二醛(MDA)、乳酸脱氢酶(LDH)水平;双荧光素酶报告实验验证ENST00000418539.1、miR-24的靶向关系;蛋白免疫印迹法检测半胱氨酰天冬氨酸特异性蛋白酶3(Caspase-3)、半胱氨酰天冬氨酸特异性蛋白酶9(Caspase-9)的表达。结果与对照组比较,模型组细胞凋亡率明显升高(P<0.05),Caspase-3、Caspase-9蛋白水平明显升高(P<0.05),MDA、LDH水平明显升高(P<0.05);与模型组、si-NC组比较,si-ENST00000418539.1组细胞凋亡率明显降低(P<0.05),Caspase-3、Caspase-9蛋白水平明显降低(P<0.05),MDA、LDH水平明显降低(P<0.05);双荧光素酶报告实验证实ENST00000418539.1可靶向结合miR-24。与si-ENST00000418539.1+anti-miR-NC组比较,si-ENST00000418539.1+anti-miR-24组细胞凋亡率明显升高(P<0.05),Caspase-3、Caspase-9蛋白水平明显升高(P<0.05),MDA、LDH水平明显升高(P<0.05)。结论干扰ENST00000418539.1表达可负向调控miR-24的表达,从而抑制H₂O₂诱导心肌细胞凋亡及减轻氧化应激损伤。     

鱼腥草提取物去甲头花千金藤二酮B对H2O2诱导的海马神经元损伤的作用及可能机制

目的探讨鱼腥草提取物去甲头花千金藤二酮B(NB)对H_2O_2诱导的海马神经元细胞损伤的作用及可能机制。方法将对数生长期的HT22海马神经细胞分为正常对照组、H_2O_2处理组、NB干预组(H_2O_2+NB)、单独NB组和阳性药物对照组(H_2O_2+NAC)。其中H_2O_2组为加入终浓度为300μmol/L的H_2O_2与细胞共孵育24 h;H_2O_2+NB组和H_2O_2+NAC组是于H_2O_2处理前2 h分别给予100μmol/L的NB或5 mmol/L的NAC共孵育24 h。为明确磷脂酰肌醇-3激酶/蛋白激酶B/血红素氧合酶1(PI3K/Akt/HO-1)途径是否参与了NB的神经保护作用,对细胞给予PI3K抑制剂Ly294002预处理30 min,再加入NB及H_2O_2共孵育24 h。CCK-8法检测细胞存活率,流式细胞仪检测细胞凋亡率,生化试剂盒检测细胞超氧化物歧化酶(SOD)、丙二醛(MDA)及谷胱甘肽(GSH)及细胞上清液乳酸脱氢酶(LDH)水平,Western blotting检测凋亡蛋白Bcl-2和Bax,及相关信号通路蛋白p-Akt和HO-1水平。采用SPSS 19.0软件进行统计分析,组间比较采用方差分析或Tukey′s检验。结果 (1)细胞活性。与空白对照组比较,H_2O_2组细胞存活率明显下降,细胞上清LDH含量显著增加,细胞形态出现明显皱缩、空泡变性及细胞减少。给予NB或NAC干预可显著增加细胞活性、减少LDH释放,细胞形态的破坏得到明显改善。(2)氧化损伤指标。与正常对照组比较,H_2O_2组细胞MDA水平显著增加,SOD及GSH活性显著降低;与H_2O_2组比较,加入NB或NAC的细胞MDA漏出量显著降低,SOD、GSH活性显著升高。(3)细胞凋亡情况。流式细胞术结果显示,与正常对照组比较,H_2O_2组细胞凋亡率明显增高,加入NB及NAC处理后,与H_2O_2组比较,细胞凋亡率显著降低(P0.05)。Western blotting结果显示,与正常对照组比较,H_2O_2处理组Bcl-2表达显著下降,Bax水平显著升高;与H_2O_2处理组比较,给予NB或阳性药物NAC处理的细胞Bcl-2显著升高,Bax水平显著下降。(4)相关信号通路蛋白表达。Western blotting显示,与正常对照组比较,H_2O_2组p-Akt、HO-1水平升高;与H_2O_2组相比,NB+H_2O_2组p-Akt、HO-1水平显著升高(P0.05)。上述所有指标在正常对照组与NB组细胞间比较差异均无统计学意义(P0.05)。另外,加入Ly294002组的细胞p-AKT及HO-1表达水平均较H_2O_2+NB组显著降低。结论 NB可通过抗氧化、抗凋亡减轻H_2O_2诱导的神经元损伤,发挥神经保护作用,其机制可能是激活PI3K/Akt/HO-1信号通路。     

H(2)O(2) regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways

Reactive oxygen species (ROS) can act as signaling molecules to stimulate either hypertrophy or apoptosis in cardiac myocytes. We tested the hypothesis that the phenotypic effects of ROS are due to differential, concentration-dependent activation of specific kinase signaling pathways. Adult rat ventricular myocytes were exposed to H(2)O(2) over a broad concentration range (10-1000 microM). Low concentrations of H(2)O(2) (10-30 microM) increased protein synthesis without affecting survival. Higher concentrations of H(2)O(2) (100-200 microM) increased apoptosis (assessed by TUNEL). Still higher concentrations of H(2)O(2) (300-1000 microM) caused both apoptosis and necrosis. A hypertrophic concentration of H(2)O(2) (10 microM) increased the activity of ERK1/2, but not that of JNK, p38 kinase or Akt. An apoptotic concentration of H(2)O(2) (100 microM) activated JNK, p38 kinase and Akt, and further activated ERK1/2. The MEK1/2 inhibitor U0126 prevented the hypertrophic effect of 10 microM H(2)O(2). The apoptotic effect of 100 microM H(2)O(2) was inhibited bya dominant-negative JNK adenovirus, and was potentiated by U0126 or an Akt inhibitor. Thus, the concentration-dependent effects of ROS on myocyte hypertrophy and growth are due, at least in part, to the differential activation of specific kinase signaling pathways that regulate hypertrophy and apoptosis.     

解偶联蛋白2过表达张氏肝细胞的构建及其对线粒体膜电位和活性氧的影响

目的 构建稳定过表达人解偶联蛋白2 (UCP2)的张氏肝细胞株,观察UCP2对线粒体膜电位(MMP)和活性氧(ROS)的作用. 方法 将含有人UCP2 cDNA全长的重组质粒(pcDNA3.1-hU CP2)转染张氏肝细胞系,pcDNA3.1空载体作为对照.Zeocin筛选稳定表达UCP2的细胞株,Western blot和免疫细胞化学鉴定UCP2蛋白表达.利用不同剂量UCP2抑制剂京尼平(25、50、100μmol/L),预处理稳定表达UCP2的细胞株.荧光分光光度法检测MMP和ROS水平变化.数据分析用单因素方差分析和q检验(Newman-keuls法),P＜0.05为差异有统计学意义.结果 pcDNA3.1-hU CP2成功转染张氏肝细胞,UCP2相对表达量约为对照组的1.6倍.过表达细胞罗丹明123和2 ′,7 ′ -二氯氢化荧光素二脂荧光强度(分别为11.11±2.76和4.97±0.62)均明显低于正常对照组张氏肝细胞(分别为15.56±2.55和6.14±1.25,q值分别为4.80和3.35,P＜0.01和P＜ 0.05)和空载体对照组肝细胞(分别为16.11±2.93和6.23±1.13,q值分别为5.40和3.60,P＜0.01和P＜ 0.05);空载体组上述两指标与正常对照组相比,差异均无统计学意义.京尼平低、中、高剂量组与过表达组相比,罗丹明123的荧光强度(分别为14.89±2.89,17.89±2.93,24.00±2.55,q值分别为4.08,7.33和13.93,P值均＜0.01)和2 ′,7 ′-二氯氢化荧光素二脂的荧光强度(分别为9.16±0.78,10.84±1.09, 11.83±1.25,q值分别为12.00,16.83和19.67,P值均＜0.01)均明显升高,并呈现剂量依赖性.结论 成功构建稳定过表达人U CP2的张氏肝细胞株,UCP2表达水平及活性变化可通过MMP和ROS影响线粒体功能.