Diazoxide triggers cardioprotection against apoptosis induced by oxidative stress

Although mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels have been reported to reduce the extent of apoptosis, the critical timing of mitoK(ATP) channel opening required to protect myocytes against apoptosis remains unclear. In the present study, we examined whether the mitoK(ATP) channel serves as a trigger of cardioprotection against apoptosis induced by oxidative stress. Apoptosis of cultured neonatal rat cardiomyocytes was determined by flow cytometry (light scatter and propidium iodide/annexin V-FITC fluorescence) and by nuclear staining with Hoechst 33342. Mitochondrial membrane potential (DeltaPsi) was measured by flow cytometry of cells stained with rhodamine-123 (Rh-123). Exposure to H(2)O(2) (500 microM) induced apoptosis, and the percentage of apoptotic cells increased progressively and peaked at 2 h. This H(2)O(2)-induced apoptosis was associated with the loss of DeltaPsi, and the time course of decrease in Rh-123 fluorescence paralleled that of apoptosis. Pretreatment of cardiomyocytes with diazoxide (100 microM), a putative mitoK(ATP) channel opener, for 30 min before exposure to H(2)O(2) elicited transient and mild depolarization of DeltaPsi and consequently suppressed both apoptosis and DeltaPsi loss after 2-h exposure to H(2)O(2). These protective effects of diazoxide were abrogated by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microM) but not by the sarcolemmal K(ATP) channel blocker HMR-1098 (30 microM). Our results suggest for the first time that diazoxide-induced opening of mitoK(ATP) channels triggers cardioprotection against apoptosis induced by oxidative stress in rat cardiomyocytes.     

Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells

Oxidative stress may cause apoptosis of cardiomyocytes in ischemia-reperfused myocardium, and heat shock pretreatment is thought to be protective against ischemic injury when cardiac myocytes are subjected to ischemia or simulated ischemia. However, the detailed mechanisms responsible for the protective effect of heat shock pretreatment are currently unclear. The aim of this study was to determine whether heat shock pretreatment exerts a protective effect against hydrogen peroxide(H2O2)-induced apoptotic cell death in neonatal rat cardiomyocytes and C2C12 myogenic cells and whether such protection is associated with decreased release of second mitochondria-derived activator of caspase-direct IAP binding protein with low pl (where IAP is inhibitor of apoptosis protein) (Smac/DIABLO) from mitochondria and the activation of caspase-9 and caspase-3. After heat shock pretreatment (42 +/- 0.3 degrees C for 1 hour, recovery for 12 hours), cardiomyocytes and C2C12 myogenic cells were exposed to H2O2 (0.5 mmol/L) for 6, 12, 24, and 36 hours. Apoptosis was evaluated by Hoechst 33258 staining and DNA laddering. Caspase-9 and caspase-3 activities were assayed by caspase colorimetric assay kit and Western analysis. Inducible heat shock proteins (Hsp) were detected using Western analysis. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blot and indirect immunofluorescence analysis. (1) H2O2 (0.5 mmol/L) exposure induced apoptosis in neonatal rat cardiomyocytes and C2C12 myogenic cells, with a marked release of Smac/DIABLO from mitochondria into cytoplasm and activation of caspase-9 and caspase-3, (2) heat shock pretreatment induced expression of Hsp70, Hsp90, and alphaB-crystallin and inhibited H2O2-mediated Smac/DIABLO release from mitochondria, the activation of caspase-9, caspase-3, and subsequent apoptosis. H2O2 can induce the release of Smac/DIABLO from mitochondria and apoptosis in cardiomyocytes and C2C12 myogenic cells. Heat shock pretreatment protects the cells against H2O2-induced apoptosis, and its mechanism appears to involve the inhibition of Smac release from mitochondria.     

Dual roles of mitochondrial K(ATP) channels in diazoxide-mediated protection in isolated rabbit hearts

Whether the mitochondrial ATP-dependent potassium (mK(ATP)) channel is the trigger or the mediator of cardioprotection is controversial. We investigated the critical time sequences of mK(ATP) channel opening for cardioprotection in isolated rabbit hearts. Pretreatment with diazoxide (100 microM), a selective mK(ATP) channel opener, for 5 min followed by 10 min washout before the 30-min ischemia and 2-h reperfusion significantly reduced infarct size (9 +/- 3 vs. 35 +/- 3% in control), indicating a role of mK(ATP) channels as a trigger of protection. The protection was blocked by coadministration of the L-type Ca(2+) channel blockers nifedipine (100 nM) or 5-hydroxydecanoic acid (5-HD; 50 microM) or by the protein kinase C (PKC) inhibitor chelerythrine (5 microM). The protection of diazoxide was not blocked by 50 microM 5-HD but was blocked by 200 microM 5-HD or 10 microM glybenclamide administrated 5 min before and throughout the 30 min of ischemia, indicating a role of mK(ATP) opening as a mediator of protection. Giving diazoxide throughout the 30 min of ischemia also protected the heart, and the protection was not blocked by chelerythrine. Nifedipine did not affect the ability of diazoxide to open mK(ATP) channels assessed by mitochondrial redox state. In electrically stimulated rabbit ventricular myocytes, diazoxide significantly increased Ca(2+) transient but had no effect on L-type Ca(2+) currents. Our results suggest that opening of mK(ATP) channels can trigger cardioprotection. The trigger phase may be induced by elevation of intracellular Ca(2+) and activation of PKC. During the lethal ischemia, mK(ATP) channel opening mediates the protection, independent of PKC, by yet unknown mechanisms.     

Influence of simulated ischemia on apoptosis induction by oxidative stress in adult cardiomyocytes of rats

Oxidative stress may cause apoptosis of cardiomyocytes in ischemic-reperfused myocardium. We investigated whether ischemia-reperfusion modifies the susceptibility of cardiomyocyte induction of apoptosis by oxidative stress. Ischemia was simulated by incubating isolated cardiomyocytes from adult rats in an anoxic, glucose-free medium, pH 6.4, for 3 h. Annexin V-fluorescein isothiocyanate/propidium iodide staining and the detection of DNA laddering were used as apoptotic markers. H(2)O(2) (7.5 micromol/l) induced apoptosis in 20.1 +/- 1.8% of cells under normoxic conditions but only 14.4 +/- 1.6% (n = 6, P < 0.05) after ischemia-reoxygenation. This partial protection of ischemic-reoxygenated cells was observed despite a reduction in their cellular glutathione content, from 11.4 +/- 1.9 in normoxic controls to 2.9 +/- 0.8 nmol/mg protein (n = 3, P < 0.05). Elevation of end-ischemic glutathione contents by pretreatment with 1 mmol/l N-acetylcysteine entirely protected ischemic-reoxygenated cells against induction of apoptosis by H(2)O(2). In conclusion, ischemia-reperfusion can protect cardiomyocytes against induction of apoptosis by exogenous oxidative stress. This endogenous protective effect is most clearly demonstrated when control and postischemic cardiomyocytes are compared at similar glutathione levels.     

Mechanism of apoptosis induced by a new topoisomerase inhibitor through the generation of hydrogen peroxide

TAS-103, a new anticancer drug, induces DNA cleavage by inhibiting the activities of topoisomerases I and II. We investigated the mechanism of TAS-103-induced apoptosis in human cell lines. Pulsed field gel electrophoresis revealed that in the leukemia cell line HL-60 and the H(2)O(2)-resistant subclone, HP100, TAS-103 induced DNA cleavage to form 1-2-Mb fragments at 1 h to a similar extent, indicating that the DNA cleavage was induced independently of H(2)O(2). TAS-103-induced DNA ladder formation in HP100 cells was delayed compared with that seen at 4 h in HL-60 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded increases in mitochondrial membrane potential (DeltaPsim) and caspase-3 activation. Inhibitors of poly(ADP-ribose) polymerase (PARP) prevented both TAS-103-induced H(2)O(2) generation and DNA ladder formation. The levels of NAD(+), a PARP substrate, were significantly decreased in HL-60 cells after a 3-h incubation with TAS-103. The decreases in NAD(+) levels preceded both increases in DeltaPsim and DNA ladder formation. Inhibitors of NAD(P)H oxidase prevented TAS-103-induced apoptosis, suggesting that NAD(P)H oxidase is the primary enzyme mediating H(2)O(2) formation. Expression of the antiapoptotic protein, Bcl-2, in BJAB cells drastically inhibited TAS-103-induced apoptosis, confirming that H(2)O(2) generation occurs upstream of mitochondrial permeability transition. Therefore, these findings indicate that DNA cleavage by TAS-103 induces PARP hyperactivation and subsequent NAD(+) depletion, followed by the activation of NAD(P)H oxidase. This enzyme mediates O(2)(-)-derived H(2)O(2) generation, followed by the increase in DeltaPsim and subsequent caspase-3 activation, leading to apoptosis.     

Leptin protects H9c2 rat cardiomyocytes from H2O2-induced apoptosis

Obesity is a known risk factor for induction of myocardial infarction, but, paradoxically, may also confer a protective effect against subsequent remodeling leading to heart failure. In this study, we investigated the effect of leptin, the product of the obese (ob) gene, on cardiomyocyte apoptosis, a well-characterized component of cardiac remodeling after myocardial infarction. Exposing H9c2 cells to H(2)O(2) decreased cell viability, and this was attenuated by pretreating cells with leptin for 1 h, but not 24 h. Leptin also attenuated the ability of H(2)O(2) to increase phosphatidylserine exposure and annexin V binding. Further investigation of underlying mechanisms of leptin's protective effect demonstrated that the H(2)O(2)-induced decrease in mitochondrial membrane potential (Psi) leading to cytochrome c release was attenuated by leptin pretreatment, and this was associated with reduced translocation of the pro-apoptotic Bax protein to the mitochondrial membrane. Finally, leptin prevented H(2)O(2)-induced increases in caspase-3 cleavage and activity, although again 24 h leptin pretreatment did not confer significant protection. In summary, we have demonstrated that acute leptin pretreatment mediates anti-apoptotic effects in H9c2 rat cardiomyocytes, which may be of significance in clarifying the direct impact of leptin on the heart.     

线粒体钾通道参与葛根素抗心肌细胞缺氧/复氧损伤的作用

目的:探讨线粒体ATP敏感性钾通道和线粒体钙激活钾通道在葛根素预处理抗心肌细胞缺氧/复氧损伤中的作用。方法:采用酶解分离大鼠心肌细胞复制心肌细胞缺氧/复氧模型,台盼蓝拒染法测定心肌细胞存活率;四甲基罗丹明乙酯(TMRE)孵育测定线粒体膜电位值;分离线粒体测定线粒体渗透性转换孔开放程度。结果:与缺氧/复氧组相比,葛根素(0.24mmol/L)预处理5min可明显增加心肌细胞的存活率,线粒体ATP敏感性钾通道抑制剂5-羟基癸酸(100μmol/L,预处理20min)或线粒体钙激活钾通道阻断剂paxilline(1μmol/L,预处理5min)均可拮抗葛根素的作用。葛根素预处理可明显减弱缺氧引起的线粒体膜电位的耗损,5-羟基癸酸和paxilline都能明显拮抗其作用。在分离心肌线粒体模型上,葛根素显著减弱CaCl2诱导的线粒体在A520处吸光度降低,其作用与单独应用线粒体渗透性转换孔抑制剂环孢菌素A相似;5-羟基癸酸和paxilline可拮抗葛根素的保护作用。结论:在大鼠分离心肌细胞模型或分离线粒体模型上,葛根素预处理具有抗缺氧/复氧损伤的作用,这种保护作用可能与其促进线粒体ATP敏感性钾通道和线粒体钙激活钾通道的开放,进而稳定线粒体膜电位,抑制线粒体渗透性转换孔开放有关。     

Sasanquasaponin up-regulates anion exchanger 3 expression and elicits cardioprotection via NO/RAS/ERK1/2 pathway

We have shown recently that sasanquasaponin (SQS) can inhibit ischemia/reperfusion-induced elevation of intracellular Cl(-) concentration ([Cl(-)](i)) and elicit cardioprotection by up-regulating anion exchanger 3 (AE(3)) expression. In the present study, we futher analysed the intracellular signal transduction pathways by which SQS up-regulates AE(3) expression and elicits cardioprotection. Cardiomyocytes were incubated for 24 h with or without 10 μmol/L SQS, followed by simulated ischemia/reperfusion (sI/R). NO formation, Ras activity, and extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation were measured appropriately. We showed that SQS pretreatment efficiently attenuated viability loss and lactate dehydrogenase leakage induced by sI/R in cardiomyocytes. Moreover, SQS induced NO production and promoted Ras activation, which futher promoted extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation. These effects were paralleled by an increase in AE(3) expression. However, when the cardiomyocytes were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (c-PTIO; an NO scavenger), S-trans-trans-farnesylthiosalicylic acid (FTS) (a Ras inhibitor), U0126 (an ERK1/2 inhibitor), respectively, the increase in AE(3) expression occurring during SQS pretreatment was almost completely abolished and, as a result, SQS-induced cardioprotection was prevented. Our findings indicate that SQS might up-regulate AE(3) expression through NO/Ras/ERK1/2 signal pathway to elicit cardioprotection in cultured cardiomyocytes.     

Protective effect of eicosapentaenoic acid on palmitate-induced apoptosis in neonatal cardiomyocytes

Long chain polyunsaturated fatty acids (PUFAs) play an important role in cardioprotection. These effects have been largely attributed to membrane docosahexaenoic acid. Conversely, saturated fatty acids trigger apoptosis in cardiomyocytes, with modifications of mitochondrial properties including cardiolipin loss, cytochrome c release and caspase-3 activation. The purpose of this study was to investigate the chronic effect of eicosapentaenoic acid (EPA) on mitochondrial apoptosis induced by palmitate treatment and the associated signalling pathways. Confluent cultures of rat neonatal cardiomyocytes were treated for 2 days in media enriched with either EPA or arachidonic acid (AA) and then exposed to palmitate (0.5 mM) to induce apoptosis, in the absence of PUFA supplements. The EPA treatment resulted in significant membrane enrichment in n-3 PUFAs, especially in docosapentaenoic acid (DPA), and a large decrease in AA. Both AA and EPA treatments prevented caspase-3 activation, translocation of Bax to the mitochondria and release of cytochrome c induced by palmitate treatment. Furthermore, EPA, but not AA prevented the loss of mitochondrial cardiolipin due to apoptosis. These results suggest that EPA supplementation is able to protect cardiomyocytes against palmitate-induced apoptosis via an implication of different mitochondrial elements, possibly through its elongation to DPA, which is very efficient in cardiomyocytes.     

一氧化氮供体对过氧化氢引起的心肌细胞损伤的保护作用

关于一氧化氮(NO)对心肌细胞是否具有保护作用目前尚存在争议,为探讨NO对过氧化氢(H2O2)引起的心肌细胞损伤是否具有保护作用及其可能的机制,实验将体外培养的新生大鼠心肌细胞分为3组(1)阴性对照组(Normal组);(2)H2O2组H2O2(0.1mmol/L)与心肌细胞共育4h;(3)S-亚硝基-N-乙酰青霉胺(SNAP)+H2O2组NO供体SNAP(0.5mmol/L)处理心肌细胞10min后,加入H2O2与心肌细胞共育4 h.用流式细胞术检测心肌细胞凋亡率,心肌细胞损伤程度以心肌细胞存活率和乳酸脱氢酶(lactate dehydrogenase,LDH)活性来表示,同时检测心肌细胞超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(MDA)含量.通过激光共聚焦显微术检测在不同处理条件下心肌细胞胞内钙的变化.结果表明,正常心肌细胞LDH活性和细胞存活率分别为631.4±75.6 U/L和93.1±6.2%,细胞凋亡率为0;H2O2处理细胞后可使细胞LDH活性显著增高(1580.5±186.7 U/L,P＜0.01),细胞存活率明显下降(58.3±7.6%,P＜0.01),流式细胞仪检测到大量心肌细胞凋亡,凋亡率为26.4±5.7%;SOD活性较正常细胞19.67±0.85 NU/ml显著下降,为14.73±1.68 NU/m(P＜0.01),MDA含量较正常细胞6.95±0.83μmol/L显著增高,为15.35±3.49μmol/L(P＜0.01).SNAP预处理细胞可显著提高心肌细胞存活率(79.7±9.3%,P＜0.01),降低LDH活性和细胞凋亡率(分别为957.8±110.9 U/L和9.1±3.3%,P＜0.01);并提高细胞抗氧化能力,表现为较H2O2处理组的SOD活性增高(21.36±3.11 NU/ml,P＜0.01),MDA含量下降(9.12±1.47 μmol/L,P＜0.01).激光共聚焦显微镜检测结果表明,H2O2可升高细胞内钙,而SNAP则可降低细胞内钙,SNAP预处理细胞后可取消H2O2升高细胞内钙的作用.上述结果提示,NO供体SNAP可对抗H2O2对心肌细胞的损伤,其机制与提高心肌细胞抗氧化损伤能力和对抗H2O2引起的细胞内钙超载有关.     

Protective effects of the potassium channel opener-diazoxide against injury in neonatal rat ventricular myocytes

The mitochondrial ATP-regulated potassium channel is present in the inner membrane of heart mitochondria. Similarly to plasma membrane K(ATP), the mitochondrial channel is inhibited by antidiabetic sulfonylureas and activated by potassium channel openers, such as diazoxide. In the present work, the cytoprotective properties of diazoxide on the H9c2 cardiac myoblast cell line and neonatal rat ventricular cardiomyocytes were analysed. It was observed that 100 micromol/l diazoxide protected neonatal rat ventricular cardiomyocytes, but not H9c2 myoblasts, against injury induced by hydrogen peroxide or simulated ischemia. Moreover, diazoxide prevented hydrogen peroxide-induced mitochondrial potential depolarisation in neonatal rat ventricular cardiomyocytes. Diazoxide, at the same time, did not affect the expression level of the anti-apoptotic protein bcl-2 in these cells. The protective effects of diazoxide were suppressed by 5-hydroxydecanoic acid, a potassium channel blocker. These observations suggest that activation of the mitochondrial ATP-regulated potassium channel plays an important role in protection of neonatal cardiomyocytes against injury.     

Oxytocin protects cardiomyocytes from apoptosis induced by ischemia-reperfusion in rat heart: role of mitochondrial ATP-dependent potassium channel and permeability transition pore

The current study examines the protective effect of oxytocin (OT) on cardiomyocyte apoptosis modulated by mitochondrial ATP-dependent potassium (mitoKATP) channel and permeability transition pore (mPTP) in the preconditioned myocardium of anesthetized rats. Eighty rats were equally divided into eight groups. The hearts of all animals except for the sham group were subjected to 25 min ischemia and 120 min reperfusion. Oxytocin, 5-hydroxydeconoate (5-HD), a specific inhibitor of the mitoKATP channel, and atractyloside (ATRC), an mPTP opener, were used prior to ischemia. Hemodynamic parameters were recorded throughout the experiment. Evaluations were made by infarct size, plasma lactate dehydrogenase level (LDH), transmission electron microscopy (TEM) and immunohistochemistry studies. OT prevented mean arterial pressure drop during early phase of ischemia and reperfusion. Treatment with OT before IR induction normalizes cardiomyocytes both in light microscopy and TEM observations. In addition, OT significantly reduced TUNEL- and increased Bcl-2-labeled positive cell number relative to IR (p<0.05). However, 5HD or ATRC inhibited the protective effects of OT on cardiomyocytes damaged by IR (p<0.05). Ultrastructural changes including extensive myofibril loss, sarcolemmal disruption and mitochondrial swelling due to amorphous dens bodies indicate necrosis induction in 5HD and ATRC as well as in IR groups. Restoration of immunohistochemistry parameters and protection against IR-induced ultrastructural changes confirm OT cardioprotective effects via mitoKATP channel and mPTP modulation in apoptosis induced by ischemia-reperfusion.     

过氧化氢预处理对抗氧化应激诱导的PC12细胞凋亡

氧化应激可明显地诱导细胞凋亡。本研究旨在探讨H2O2预处理能否对H2O2诱导的PC12细胞凋亡生产保护作用及ATP敏感性K^ （ATP-sensitive potassinm,KATP）通道在其中的作用。采用PI染色流式细胞仪（flow cytometry, FCM）检测PC12细胞凋亡。结果表明,经10μmol/L H2O2预处理90min的PC12细胞,分别在20、30、50和100μmol/L H2O2作用24h后,其细胞凋亡率明显下降,与未经H2O2的预处理的PC12细胞相比,差异极显著（P＜0.01）,表明H2O2预处理对H2O2诱导PC12细胞凋亡具有保护作用。用10μmol/L的KATP通道激动齐pinacidil(Pin)可显著减少30和50μmol/L H2O2诱导的PC12细胞凋亡,10μmol/L的KATP通道拮抗齐glybenclamide（Gly）则可显著地抑制甚至取消KATP通道激动剂Pin对H2O3诱导PC12细胞凋亡的保护作用,但并不影响H2O2预处理对H2O2诱导PC12细胞凋亡的保护作用;然而,当联合应用H2O2预处理与Pin时,对PC12细胞凋亡的保护作用显大于各自的细胞凋亡作用。提示KATP通道开放不仅对H2O2诱导PC12细胞凋亡具有保护作用,而且与H2O2预处理一起产生抗PC12细胞凋亡的协同作用。但KATP通道开放可能不参与H2O2预处理的适应性保护作用。     

Bradykinin induces mitochondrial ROS generation via NO,cGMP, PKG,and mitoKATP channel opening and leads to cardioprotection

Bradykinin (BK) mimics ischemic preconditioning by generating reactive oxygen species (ROS). To identify intermediate steps that lead to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker Red stain, which becomes fluorescent after exposure to ROS. Fluorescence intensity in treated cells was expressed as a percentage of that in paired, untreated cells. BK (500 nM) caused a 51 +/- 16% increase in ROS generation (P < 0.001). Coincubation with either the BK B2-receptor blocker HOE-140 (5 microM) or the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) prevented this increase, which confirms that the response was receptor mediated and ROS were actually being measured. Closing mitochondrial ATP-sensitive K+ (mitoKATP) channels with 5-hydroxydecanoate (5-HD, 1 mM) prevented increased ROS generation. BK-induced ROS generation was blocked by Nomega-nitro-m-arginine methyl ester (m-NAME, 200 microM), which implicates nitric oxide as an intermediate. Blockade of guanylyl cyclase with 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10 microM) aborted BK-induced ROS generation but not that from diazoxide, a direct opener of mitoKATP channels. The protein kinase G (PKG) blocker 8-bromoguanosine-3',5'-cyclic monophosphorothioate (25 microM) eliminated the effects of BK. Conversely, direct activation of PKG with 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (100 microM) increased ROS generation (39 +/- 15%; P < 0.004) similar to BK. This increase was blocked by 5-HD. Finally, the nitric oxide donor S-nitroso-N-acetylpenicillamine (1 microM) increased ROS by 34 +/- 6%. This increase was also blocked by 5-HD. In intact rabbit hearts, BK (400 nM) decreased infarction from 30.5 +/- 3.0 of the risk zone in control hearts to 11.9 +/- 1.4% (P < 0.01). This protection was aborted by either 200 microM m-NAME or 2 microM ODQ (35.4 +/- 5.7 and 30.4 +/- 3.0% infarction, respectively; P = not significant vs. control). Hence, BK preconditions through receptor-mediated production of nitric oxide, which activates guanylyl cyclase. The resulting cGMP activates PKG, which opens mitoKATP. Subsequent release of ROS triggers cardioprotection.     

Mechanism for generation of hydrogen peroxide and change of mitochondrial membrane potential during rotenone-induced apoptosis

Rotenone, an inhibitor of NADH dehydrogenase complex, is a naturally occurring insecticide, which is capable of inducing apoptosis. Rotenone-induced apoptosis is considered to contribute to its anticancer effect and the etiology of Parkinson's disease (PD). We demonstrated that rotenone induced internucleosomal DNA fragmentation, DNA ladder formation, in human cultured cells, HL-60 (promyelocytic leukemia) and BJAB cells (B-cell lymphoma). Flow cytometry showed that rotenone induced H2O2 generation, followed by significant changes in the mitochondrial membrane potential (DeltaPsim). Caspase-3 activity increased in HL-60 cells in a time-dependent manner. These apoptotic events were delayed in HP100 cells, an H2O2-resistant clone of HL-60, confirming the involvement of H2O2 in apoptosis. Expression of anti-apoptotic protein, Bcl-2, in BJAB cells drastically inhibited DeltaPsim change and DNA ladder formation but not H2O2 generation, confirming the participation of mitochondrial dysfunction in apoptosis. NAD(P)H oxidase inhibitors prevented H2O2 generation and DNA ladder formation. These results suggest that rotenone induces O2(-)-derived H2O2 generation through inhibition of NADH dehydrogenase complex and/or activation of NAD(P)H oxidase, and H2O2 generation causes the disruption of mitochondrial membrane in rotenone-induced apoptosis.     

MitoQ对高糖诱导的心肌细胞线粒体功能影响

目的:探讨MitoQ对高糖诱导的心肌细胞线粒体功能影响。方法:常规获取与纯化SD大鼠新生仔鼠心肌细胞,分为对照组、高糖组、实验组。对照组用含10%血清的DMEM培养基(5.5 mmol/L葡萄糖)培养;高糖组用含血清的高糖DMEM培养基(33mmol/L葡萄糖)培养;实验组用含血清的高糖DMEM培养基(33 mmol/L葡萄糖)和MitoQ。MTT法检测心肌细胞存活率,氯离子荧光探针检测细胞内氯离子浓度,流式细胞术检测各组心肌细胞凋亡率,超氧化物阴离子荧光染色检测心肌细胞活性氧(reactive oxygen,ROS)含量,利用ATP检测试剂盒检测心肌细胞中的ATP水平,Western blot法检测心肌细胞胱天蛋白酶3(caspase-3)蛋白水平。结果:与对照组相比,高糖组的心肌细胞增凋亡率、ROS产生、氯离子相对浓度均明显增加,ATP显著降低(P0.05),细胞内caspase-3蛋白表达显著上升(P0.05);与高糖组相比,实验组凋亡率降低,ROS产生、细胞内caspase-3蛋白表达均显著降低(P0.05)。结论:高糖会引起心肌细胞线粒体障碍,造成心肌细胞凋亡,MitoQ可降低细胞内ROS和caspase-3水平,抑制心肌细胞凋亡,改善心肌细胞线粒体功能。     

Schisandrin B stereoisomers protect against hypoxia/reoxygenation-induced apoptosis and inhibit associated changes in Ca2+-induced mitochondrial permeability transition and mitochondrial membrane potential in H9c2 cardiomyocytes

The effects of schisandrin B stereoisomers, (+/-)gamma-schisandrin [(+/-)gamma-Sch] and (-)schisandrin B [(-)Sch B], on hypoxia/reoxygenation-induced apoptosis were investigated in H9c2 cardiomyocytes. Changes in cellular reduced glutathione (GSH) levels, Ca(2+)-induced mitochondrial permeability transition (MPT), and mitochondrial membrane potential (Deltapsi(m)) values, were examined in (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells, without or with hypoxia/reoxygenation challenge. The (+/-)gamma-Sch and (-)Sch B (2.5-5.0 microM) pretreatments protected against hypoxia/reoxygenation-induced apoptosis of H9c2 cells in a concentration-dependent manner, with (-)Sch B being more potent. The degrees of protection decreased, however, at the higher drug concentrations of 7.5 microM in both (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells. The anti-apoptotic effects of the drugs were further evidenced by the suppression of hypoxia/reoxygenation-induced mitochondrial cytochrome c release and the subsequent cleavage of caspase 3 and poly-ADP-ribose polymerase after (-)Sch B pretreatment. Both (+/-)gamma-Sch and (-)Sch B pretreatments increased GSH levels in H9c2 cells, with (-)Sch B being more potent. Hypoxia/reoxygenation challenge caused a depletion in cellular GSH and the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B was associated with enhancement of cellular GSH in H9c2 cells, as compared to the drug-unpretreated control. Whereas hypoxia/reoxygenation challenge increased the extent of Ca(2+)-induced MPT pore opening and decreased Deltapsi(m) in H9c2 cardiomyocytes, cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by (+/-)gamma-Sch/(-)Sch B pretreatments was associated with a decreased sensitivity to Ca(2+)-induced MPT and an increased Deltapsi(m) in both unchallenged and challenged cells, as compared to the respective drug-unpretreated controls. The degrees of protection against apoptosis correlated negatively with the extents of Ca(2+)-induced MPT (r=-0.615, P<0.01) and positively with the values of Deltapsi(m) (r=0.703, P<0.01) in (+/-)gamma-Sch/(-)Sch B-pretreated and hypoxia/reoxygenation challenged cells. The results indicate that (+/-)gamma-Sch/(-)Sch B pretreatment protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes and that the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B may at least in part be mediated by a decrease in cellular sensitivity to Ca(2+)-induced MPT, which may in turn result from enhancement of cellular GSH levels by drug pretreatments.     

Protective effects of cynaroside against H₂O₂-induced apoptosis in H9c2 cardiomyoblasts

Flavonoids with potent anti-oxidative effects are the major effective components in traditional herbal medicine used in treating cardiovascular diseases. Cynaroside is a flavonoid compound that exhibits anti-oxidative capabilities. However, little is known about its effect on oxidative injury to cardiac myocytes and the underlying mechanisms. This study was designed to investigate the protective effects of cynaroside against H(2) O(2) -induced apoptosis in H9c2 cardiomyoblasts. H9c2 cells were pretreated with cynaroside for 4 h before exposure to 150 μM H(2) O(2) for 6 h. H(2) O(2) treatment caused severe injury to the H9c2 cells, which was accompanied by apoptosis, as revealed by analysis of cell nuclear morphology, through Annexin V FITC/PI staining and caspase proteases activation. Cynaroside pretreatment significantly reduced the apoptotic rate by enhancing the endogenous anti-oxidative activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting intracellular reactive oxygen species (ROS) generation. Moreover, cynaroside moderated H(2) O(2) -induced disruption of mitochondrial membrane potential, increased the expression of anti-apoptotic protein Bcl-2 while decreased the expression of pro-apoptotic protein Bax, and thereby inhibited the release of apoptogenic factors (cytochrome c and smac/Diablo) from mitochondria in H9c2 cells. Our data also demonstrated that cynaroside pretreatment showed an inhibitory effect on the H(2) O(2) -induced increase in c-Jun N-terminal kinase (JNK) and P53 protein expression. These results suggest that cynaroside prevents H(2) O(2) -induced apoptosis in H9c2 cell by reducing the endogenous production of ROS, maintaining mitochondrial function, and modulating the JNK and P53 pathways.     

Smac/DIABLO在过氧化氢所致C2C12肌原细胞凋亡中的作用

为探讨Smac/DIABLO在过氧化氢(H₂O₂）所致C₂C₁₂肌原细胞凋亡中的作用,采用Hoechst 33258染色,观察H₂O₂ (0.5 mmol/L)处理C₂C₁₂肌原细胞不同时间后,细胞核形态学改变并计算凋亡核百分率,DNA抽提及琼脂糖电泳观察凋亡特征性梯状带,利用细胞成分分离后蛋白质印迹分析H₂O₂是否导致Smac/DIABLO从线粒体释放,采用Caspase检测试剂盒及蛋白质印迹分析Caspase-3和Caspase-9的活化,转染Smac/DIABLO基因,观察Smac/DIABLO过表达对H₂O₂所致的C₂C₁₂肌原细胞凋亡的影响.结果表明:H₂O₂处理1 h后,Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放入胞浆,2 h更明显;H₂O₂处理4 h后,Caspase-3和Caspase-9活化,12 h达高峰;H₂O₂处理24 h后,C₂C₁₂肌原细胞显示特征性的凋亡形态改变,凋亡核百分率明显升高,DNA电泳出现明显“梯状”条带.与单纯过氧化氢损伤组相比,Smac/DIABLO高表达的C₂C₁₂肌原细胞经过氧化氢损伤组的Caspase-3和Caspase-9的活化、凋亡核百分率的升高、“梯状”条带的出现均更明显.结果表明,H₂O₂可导致Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放,促进Caspase-9和Caspase-3的活化而促进细胞凋亡的发生.     

Doxorubicin-induced cardiomyocyte apoptosis: Role of mitofusin 2

BackgroundDoxorubicin (DOX) is an anti-tumor agent that is widely used in clinical setting for cancer treatment. The application of the DOX, however, is limited by its cardiac toxicity which can induce heart failure through an undefined mechanism. Mitofusin 2 (Mfn2) is a mitochondrial GTPase fusion protein that is located on the outer membrane of mitochondria (OMM). The Mfn2 plays an important role in mitochondrial fusion and fission. The aim of this study is to identify the role of the Mfn2 in DOX-induced cardiomyocyte apoptosis.MethodsCultured neonatal rat cardiomyocytes were used in this study. Mfn2 expression in cardiomyocytes was determined after the cardiomyocytes were challenged with DOX. Cardiomyocyte mitochondrial fission, mitochondrial reactive oxygen species (ROS) production was assessed with mitochondrial fragmentation and MitoSOX fluorescence probe, respectively. Cardiomyocyte apoptosis was determined with caspase3 activity and TUNEL staining.ResultsChallenging of the cardiomyocytes with DOX resulted in increasing in cardiomyocyte oxidative stress and apoptosis. In addition, levels of Mfn2 in cardiomyocytes were decreased after the cells were challenged with DOX which was associated with increased mitochondrial fission (fragmentation) and mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 attenuated the DOX-induced increase in mitochondrial fission and prevented cardiomyocyte mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 or pretreatment of cardiomyocytes with an anti-oxidant, Mito-tempo, also prevented the DOX-induced cardiomyocyte apoptosis.ConclusionOur results indicate that DOX results in a decreased cardiomyocyte Mfn2 expression which promotes mitochondrial fission and ROS production further leads to cardiomyocyte apoptosis.