Differential effects of calpain inhibitors on hypertrophy of cardiomyocytes

Two inhibitors of the calcium-dependent cysteine protease, calpain, have markedly different effects on the extent of hypertrophy induced by the alpha-adrenergic agonist, phenylephrine, of cultured neonatal rat ventricular myocytes. E64c, an inhibitor of calpain and other cysteine proteases, stimulated the hypertrophy by 59%. PD 150606, a specific calpain inhibitor, reduced the hypertrophy by 38%. Phenylephrine decreased the proteolysis of a calpain substrate by the cells 1–2 h after its addition but not at 24 h. PD 150606 inhibited proteolytic activity at all times, and the combination of phenylephrine and PD 150606 did not give greater inhibition. This suggests that cysteine proteases of the papain sub-family are involved with the hypertrophic response at two points, promoting hypertrophy at the first and limiting it at the second. Calpain appears to be the protease involved at the first point, and there may be another cysteine protease acting at the second site.     

银杏叶提取物和槲皮素对心肌细胞肥大的防治作用及其机制

目的：探讨银杏叶提取物（EGb）及其单体槲皮素（Que）对心肌细胞肥大的防治作用及其机制。方法：采用血管紧张素Ⅱ（AngⅡ）诱导新生大鼠心肌细胞肥大模型;分别在培养液中加入EGb（40／μg／ml）或Que（4／μg／m1）,观察Lowry法测定心肌细胞蛋白质含量的变化;测定SOD活性和MDA含量观察心肌细胞氧自由基代谢的变化;Western blot方法检测心肌细胞p-ERK1／2、p-JNK和p-P38蛋白表达;应用RT-PCR法检测心肌细胞c-fos mRNA表达。结果：①EGb和Que能明显抑制AngⅡ引起的心肌细胞总蛋白质含量的增加;②EGb和Que可显著提高SOD活性,降低MDA含量;③AngⅡ诱导的心肌细胞p-ERK1／2、p-JNK和p-P38表达均明显增强,Que能明显抑制AngⅡ诱导的p-JNK表达;④EGb、Que、可降低AngⅡ诱导心肌细胞c-fos mRNA表达的上调水平。结论：EGb和Que对AngⅡ诱导的心肌细胞肥大有明显的防治作用,Que的作用机制可能与ROS／JNK／c-fos信号通路有关。氧化应激参与了心肌肥大的发生发展过程。     

Hydrogen peroxide－induced changes in intracellular pH of guard cells precede stomatal closure

INTRODUCTIONEven under optimal conditions, many metabolicprocesses, including chloroplastic, mitochondrial,and plasma membrane-linked electron transportsystems, produce reactive oxygen species (ROS)such as the superoxide radical (OZ--), hydrogenperoxide (HZOZ), and the hydroxyl free radical(OH--)[1, 2]. Furthermore, the imposition of bioticand abiotic stress conditions can give rise to ex-cess concentrations of ROS, resulting in oxidativedamage at the cellular level. Interestingly, R…     

H2S对大鼠心肌肥大与miRNA-133a和Ca2+/CaN/NFATc4信号通路的影响

目的：研究硫化氢（H₂S）对心肌细胞肥大的负性调控作用与miRNA-133a介导Ca²⁺/CaN/NFATc4信号通路的关系。方法：异丙肾上腺素（ISO）诱导体外培养的大鼠心肌细胞肥大模型;Leica图像分析软件测量心肌细胞表面积;qRT-PCR检测脑钠尿肽（BNP）、β-肌球蛋白重链（β-MHC）、H₂S合酶（CSE）、miRNA-133a和钙调神经磷酸酶（CaN） mRNA表达;Western blot检测CaN、活化T细胞核因子c4（NFATc4）蛋白表达;Elisa方法检测心肌细胞H₂S含量;激光共聚焦显微镜检测心肌细胞钙离子浓度;细胞免疫荧光检测NFATc4核转位变化。结果：①心肌细胞肥大时,CSE/H₂S水平、miRNA-133a mRNA表达均显著下降。应用NaHS预处理,能上调心肌细胞CSE/H₂S水平,增加H₂S含量和miRNA-133a mRNA表达,并明显抑制心肌细胞肥大。②心肌细胞肥大时,细胞内钙离子浓度明显增加,CaN表达和NFATc4胞核蛋白表达增加,NFATc4核转位明显增强;应用NaHS预处理能明显抑制ISO诱导的上述效应。③应用antagomir-133a能逆转H₂S抑制心肌细胞肥大的作用,使心肌细胞内钙离子浓度、CaN表达和NFATc4胞核蛋白表达增加,NFATc4核转位增强。结论：H₂S通过负性调控作用抑制心肌细胞肥大,该作用可能与H₂S上调miRNA-133a的表达,抑制其下游的Ca²⁺/CaN/NFATc4信号通路的激活有关。     

Early administration of nifedipine protects against angiotensin II‐induced cardiomyocyte hypertrophy through regulating CaMKII–SERCA2a pathway and apoptosis in rat cardiomyocytes

The calcium channel blocker (CCB), nifedipine, is a more effective treatment for early‐ than late‐stage cardiac hypertrophy. We investigated the effects of early‐ and late‐stage nifedipine administration on calcium homeostasis, CaMKII (Ca²⁺/calmodulin‐dependent protein kinase II) activity and apoptosis of cardiomyocytes under hypertrophic stimulation with angiotensin II (AngII). Primary rat cardiomyocytes were divided into five treatment groups: AK, AngII plus the CaMKII inhibitor, KN‐93; AN‐1 (early‐stage), AngII plus nifedipine × 48 h; AN‐2 (late‐stage), AngII × 48 h, then AngII plus nifedipine × 48 h; C, untreated; and A, AngII × 48 h. The t1/2β [time required for intracellular Ca²⁺ concentration ([Ca²⁺]i) to decline to one half of the peak value] decreased; however, CaMKII and SERCA2a (sarcoplasmic reticulum Ca²⁺‐ATPase 2a) activities increased in the AN‐1 group compared with the AK group. In the AN‐2 group compared with the AN‐1 group, CaMKII activity, t1/2α [time required for [Ca²⁺]i to increase from the bottom to one half of peak value], t1/2β, and apoptosis increased. These results indicate that the timing of CCB administration affects the calcium concentration and apoptosis of hypertrophic cardiomyocytes through the CaMKII–SERCA2a signalling pathway, thereby influencing the drug's protective activity against cardiomyocyte hypertrophy. Copyright © 2016 John Wiley & Sons, Ltd.     

Increased intracellular [dATP] enhances cardiac contraction in embryonic chick cardiomyocytes

Although ATP is the physiological substrate for cardiac contraction, cardiac contractility is significantly enhanced in vitro when only 10% of ATP substrate is replaced with 2'-deoxy-ATP (dATP). To determine the functional effects of increased intracellular [dATP] ([dATP](i)) within living cardiac cells, we used hypertonic loading with varying exogenous dATP/ATP ratios, but constant total nucleotide concentration, to elevate [dATP](i) in contractile monolayers of embryonic chick cardiomyocytes. The increase in [dATP](i) was estimated from dilution of dye added in parallel with dATP. Cell viability, average contractile amplitude, rates of contraction/relaxation, spontaneous beat frequency, and Ca2+ transient amplitude and kinetics were examined. At total [dATP](i) above approximately 70 microM, spontaneous contractions ceased, and above approximately 100 microM [dATP](i), membrane blebbing was also observed, consistent with apoptosis. Interestingly, [dATP](i) of approximately 60 microM ( approximately 40% increase over basal [dATP](i) levels) enhanced both amplitude of contraction and the rates of contraction and relaxation without affecting beat frequency. With total [dATP](i) of approximately 60 microM or less, we found no significant change in Ca2+ transients. These data indicate that there is an "optimal" concentration of exogenously loaded [dATP](i) that under controlled conditions can enhance contractility in living cardiomyocytes without affecting beat frequency or Ca2+ transients.     

Insulin-like growth factor-II induces hypertrophy of adult cardiomyocytes via two alternative pathways

Insulin-like growth factor (IGF)-II is known to induce hypertrophy of isolated adult rat ventricular cardiomyocytes cultured in the absence of serum. However, it is not known how the growth factor exerts this hypertrophic effect. We show here that IGF-II induces hypertrophy of the cultured cardiomyocytes via two alternative pathways: (1) an IGF-I receptor-dependent pathway, or (2) a lysosome-dependent pathway when the IGF-I receptor-dependent pathway is blocked.     

八肽胆囊收缩素激活钙离子通道和酪氨酸激酶诱导豚鼠心肌细胞内的游离钙增高

探讨八肽胆囊收缩素(CCK-8)对豚鼠单个心肌细胞内游离钙浓度([Ca2+]i的影响及其信号转导机制.Fluo 3-AM标记酶消化法分离的单个心室肌细胞,用激光共聚焦显微镜测定细胞内[Ca2+]i的浓度.[Ca2+]i的变化用荧光强度(Fi)和相对荧光强度(Fi/F0%)表示.实验结果如下(1)在含Ca2+1.0 mmol/L的Tyrode's液中,CCK-8(1～104pmoVL)均可引起[Ca2+]i快速显著上升(P＜0.01).(2)用钙离子鳌合剂EGTA(3 mmol/L)和钙离子通道阻断剂nisoldipine(0.5μmol/L)预孵育心肌细胞5 min,CCK-8(102pmol/L)仅可引起[Ca2+]i缓慢轻度上升(P＜0.01).(3)用非选择性CCK受体拮抗剂丙谷胺(proglumide 6μmo1/L)或酪氨酸激酶抑制剂genistein(1 μmol/L)预孵育心肌细胞5 min,则完全抑制CCK-8诱导的[Ca2+]i升高(P＜0.01).CCK-8可通过激活其受体控制的Ca2+通道,引起Ca2+内流,诱导细胞内Ca2+释放,引起豚鼠单个心肌细胞内[Ca2+]i上升,此作用可能由酪氨酸激酶介导.     

Caspase 3 inhibition attenuates hydrogen peroxide-induced DNA fragmentation but not cell death in neuronal PC12 cells

Exposure of neurons to H(2)O(2) results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H(2)O(2)-mediated cell death is unknown. We examined H(2)O(2)-induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H(2)O(2)-scavenging enzyme catalase on this process. H(2)O(2) caused cell death in a time- and concentration-dependent manner. Cell death induced by H(2)O(2) was found to be mediated in part through an apoptotic pathway as H(2)O(2)-treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H(2)O(2) also triggered activation of caspase 3. Genetic up-regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H(2)O(2) it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H(2)O(2)-mediated cellular toxicity. These results suggest that DNA fragmentation induced by H(2)O(2) is attributable to caspase 3 activation and that H(2)O(2) may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H(2)O(2)-mediated cell death.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H₂O₂) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca²⁺/Mg²⁺-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca²⁺/Mg²⁺-supplemented medium. In Ca²⁺/Mg²⁺-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 µm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H₂O₂ (84.40±0.50 ng/egg) when compared to control eggs (80.46±1.34 ng/egg). The higher concentration of calcium ionophore (1.6 µm) induced apoptosis and pronounced generation of intracellular H₂O₂ (92.43±0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H₂O₂ level (81.20±1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H₂O₂ in rat eggs.     

蛋白激酶C对自发性高血压大鼠肥大心肌细胞无负荷收缩功能的调节

在慢性压力超负荷引起心肌肥大过程中,蛋白激酶C(protein kinase C,PKC)的激活起关键性作用,激活的PKC也能调节心肌收缩性能.本文旨在研究自发性高血压大(spontaneously hypertensive rat,SHR)心肌肥大的不同阶段PKC调节心肌收缩性能的特征.采用胶原酶法分离4月龄与10月龄Wistar-Kyoto(WKY)、SHR大鼠的心肌细胞,观测单个心肌细胞无负荷缩短幅值以及在PKC激动剂与抑制剂作用下心肌收缩性能的变化.结果表明:刺激频率从1 Hz增至3 Hz,WKY大鼠心肌细胞无负荷缩短幅值逐渐增加,呈正阶梯效应;4月龄SHR大鼠心肌细胞的缩短幅值较WKY大鼠增强,但在各刺激频率下其缩短幅值基本保持不变;10月龄SHR大鼠心肌细胞的缩短幅值在1 Hz刺激条件下与WKY大鼠无差别,随刺激频率增加,缩短幅值降低,呈负阶梯效应.在PKC激动剂PMA灌流条件下,50、100与200 nmol/L的PMA分别降低WKY大鼠心肌细胞缩短幅值至(69.8±1.9)%、(58.2 2.2)%与(22.7±2.5)%(均P<0.01),呈浓度依赖关系;PMA对4月龄SHR大鼠心肌细胞缩短幅值的降低更明显,分别降至(6.1±0.7)%、(2.4±0.2)%与(12.5±2.6)%(均P<0.01);PMA降低10月龄SHR大鼠心肌细胞缩短幅值至(65.7±1.6)%、(53.9±4.0)%与(16.3±2.0)%(均P<0.01),小于对4月龄SHR大鼠心肌细胞缩短幅值的作用.PKC抑制剂staurosporine增加WKY大鼠心肌细胞缩短幅值,在200 nmol/L的staurosporine灌流条件下,WKY大鼠、4月龄SHR大鼠、10月龄SHR大鼠心肌细胞缩短幅值分别增JJH(63.63±4.53)%、(80.82±4.61)%、(80.97±4.59)%(均P<0.05).结果提示,在SHR大鼠心肌肥大初期,具有负性肌力作用的PKC异构体可能被激活,并参与对心肌收缩性能的调节;而心肌肥大稳定阶段,这些PKC活性可能恢复至正常水平.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Attenuated SAG expression exacerbates 4-hydroxy-2-nonenal-induced apoptosis and hypertrophy of H9c2 cardiomyocytes

Abstract

Oxidative stress, associated with the accumulation of reactive oxygen species (ROS), results in numerous and detrimental effects on the myocardium such as the induction of apoptotic cell death, hypertrophy, fibrosis, dysfunction, and dilatation. The product of sensitive to apoptosis gene (SAG) is a RING finger protein that has been shown to have a protective effect against apoptosis induced by oxidative stress in various cell types. The major reactive aldehydic product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), is believed to be largely responsible for cytopathological effects observed during oxidative stress. In the present study, we showed that the transfection of H9c2 clonal myoblastic cells with small interfering RNA (siRNA) specific for SAG markedly attenuated SAG expression and exacerbates HNE-induced apoptosis and hypertrophy. The knockdown of SAG expression resulted in the modulation of cellular redox status, mitochondrial function, and cellular oxidative damage. Taken together, our results showed that the suppression of SAG expression by siRNA enhanced HNE-induced apoptosis and hypertrophy of cultured cardiomyocytes via the disruption of the cellular redox balance. Given the importance of the SAG protein in the regulation of the redox status of cardiomyocytes, we conclude that this protein may be a potential new target in the development of therapeutic agents for the prevention of cardiovascular diseases.     

Aqueous extract of the Chinese medicine, Danggui-Shaoyao-San, inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade

Danggui-Shaoyao-San (DSS), a traditional Chinese medicine used for centuries for the enhancement of women's health, has been shown to display therapeutic efficacy on senile dementia. In the present study, using a rat pheochromocytoma (PC12) cell line, the effect of DSS on hydrogen peroxide (H2O2) induced apoptosis was studied. The apoptosis in H2O2-induced PC12 cells was accompanied by downregulation of Bcl-2, upregulation of Bax, the release of mitochondrial cytochrome c into cytosol, and sequential activation of caspase-9 and -3. DSS was able to suppress all these changes and eventually protected against H2O2-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with DSS can block H2O2-induced apoptosis by the regulation of Bcl-2 family members, as well as suppression of cytochrome c release and caspase cascade activation.     

植物叶片中过氧化氢含量测定方法的改进

Ti（Ⅳ）-H₂O₂比色法因背景物质干扰而测得的植物叶片内H₂O₂含量偏高，5％三氯乙酸抽提，活性炭脱色，Ti（Ⅳ）-4-(2-吡啶偶氮)间苯二酚(PAR)比色法测得的H₂O₂含量偏低.萃取法有效地脱去丙酮提液中的色素，且H₂O₂的回收率在95％以上.用过氧化氢酶(CAT）处理作空白对照，利用H₂O₂与Ti（Ⅳ）-PAR的显色反应，建立了一种简便、快速、准确的植物叶片内的H₂O₂含量测定方法，H₂O₂的最低检测浓度为0.25 μmol·L^－1.用该方法测得多种植物叶片中H₂O₂的含量在0.1～0.8 μmol·g^－1.     

U50488H、哌唑嗪及心得安对去甲肾上腺素诱导心肌肥大的作用比较

目的：观测κ阿片受体激动对去甲肾上腺素诱导心肌肥大的抑制作用,并与哌唑嗪、心得安作用进行比较。方法：结晶紫染色法测心肌细胞增殖程度;Lowry法测心肌细胞蛋白含量;计算机图象分析系统测心肌细胞体积;[3H]-亮氨酸掺入法测心肌细胞蛋白合成。结果：①低血清环境下,NE明显诱导心肌细胞蛋白含量、蛋白合成及体积的增加,但对增殖无影响。②哌唑嗪和心得安单独作用部分抑制NE诱导的心肌肥大;联合作用则完全抑制。③U50488H明显抑制NE诱导的心肌肥大;其抑制程度与哌唑嗪和心得安联合作用类似,明显高于二者单独作用。结论：NE通过激动α1-和β-肾上腺受体途径诱导心肌肥大。κ阿片受体激动显著抑制NE诱导的心肌肥大,这可能与干预α1-AR和β-AR途径有关。     

Graphene‐amplified electrogenerated chemiluminescence of CdTe quantum dots for H2O2 sensing

Electrogenerated chemiluminescence (ECL) of thiol‐capped CdTe quantum dots (QDs) in aqueous solution was greatly enhanced by PDDA‐protected graphene (P‐GR) film that were used for the sensitive detection of H₂O₂. When the potential was cycled between 0 and ?2.3 V, two ECL peaks were observed at ?1.1 (ECL‐1) and ?1.4 V (ECL‐2) in pH 11.0, 0.1 M phosphate buffer solution (PBS), respectively. The electron‐transfer reaction between individual electrochemically‐reduced CdTe nanocrystal species and oxidant coreactants (H₂O₂ or reduced dissolved oxygen) led to the production of ECL‐1. While mass nanocrystals packed densely in the film were reduced electrochemically, assembly of reduced nanocrystal species reacted with coreactants to produce an ECL‐2 signal. ECL‐1 showed higher sensitivity for the detection of H₂O₂ concentrations than that of ECL‐2. Further, P‐GR film not only enhanced ECL intensity of CdTe QDs but also decreased its onset potential. Thus, a novel CdTe QDs ECL sensor was developed for sensing H₂O₂. Light intensity was linearly proportional to the concentration of H₂O₂ between 1.0 × 10^?5 and 2.0 x 10^‐7 mol L^?1 with a detection limit of 9.8 x 10^?8 mol L^?1. The P‐GR thin‐film modified glassy carbon electrode (GCE) displayed acceptable reproducibility and long‐term stability. Copyright © 2012 John Wiley & Sons, Ltd.     

Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H₂O₂)-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H₂O₂-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H₂O₂-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H₂O₂-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.     

A near‐infrared fluorescent probe based on boric acid hydrolysis for hydrogen peroxide detection and imaging in HeLa cells

Using the characteristics of hydrogen peroxide that are able to cleave phenyl‐boric acid selectively and efficiently, we here report a dicyanoisophorone‐boric acid ( DCP‐BA )‐based near‐infrared (NIR) fluorescent probe for detection of hydrogen peroxide. This probe shows a rapid, highly selective, and sensitive detection process for hydrogen peroxide with a significant NIR fluorescent turn‐on response that has been successfully applied to detect exogenous hydrogen peroxide in HeLa cells.