Mitochondria-assisted cell suicide: a license to kill

Although the mechanisms that underlie cardiac cell death remain cryptic, there is emerging evidence that mitochondria may play a pivotal role in this process. The mitochondrion initially deemed the "power house " is now considered to be a central integration site for biological signals that promote cell life or cell death. Since mitochondria contain the necessary apoptotic machinery to activate the cell-death pathway, it is now appreciated that mitochondria play a key decision-making role in whether a cell will live or die following a noxious signal-literally a "license to kill ". Permeability changes to the outer mitochondrial membrane, collapse of membrane potential, permeability pore complex assembly, release of cytotoxic proteins and caspase activation are associated with the mitochondrial-death pathway. Members of the Bcl-2 gene family can promote or suppress cell death by modulating mitochondrial function. Activation of the mitochondrial-death pathway has been reported in several cardiac pathologies and believed to account for the reported apoptosis observed in these disease entities. Given the meager and limited ability of cardiac muscle for repair or self-renewal after injury, the inordinate loss of cardiac cells is considered to be a key underlying factor in ventricular remodeling and decline in ventricular performance in patients with ischemic heart disease or post-myocardial infarction. This review will provide mechanistic insight into the involvement and contribution of the mitochondrion as a regulator of cell death in health and disease with particular focus on the heart.     

硫酸奎尼丁对乳大鼠心室肌细胞Kv4．3基因mRNA水平表达的影响

目的探讨硫酸奎尼丁对乳大鼠心室肌细胞Kv4．3基因mRNA水平表达的影响及其治疗Brugada综合征的分子机制。方法将乳大鼠心室肌细胞随机分为对照组及硫酸奎尼丁干预组,培养至24、48、72h,应用半定量逆转录一聚合酶链反应（RT-PCR）技术检测Kv4．3基因mRNA表达的变化。结果于预至24h对Kv4．3基因mRNA水平表达无明显影响;至48h,各干预组较对照组显著降低（P〈0．05）,且5与10μmol／L组较1μmol／L组下调作用更为显著（P〈0．05）;至72h,各干预组较对照组显著降低（P〈0．05）,而各干预组间差异无统计学意义。结论硫酸奎尼丁可显著下调Kv4-3基因mRNA水平表达,且较长时间干预时低浓度与较高浓度下调的幅度相近。     

Suppression of dynamic Ca(2+) transient responses to pacing in ventricular myocytes from mice with genetic calmodulin kinase II inhibition

The multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) is important for regulating L-type Ca(2+) current (I(Ca)) and cytoplasmic Ca(2+) (Ca(2+)(i)) uptake and release from the sarcoplasmic reticulum (SR), key elements of the 'Ca(2+)-induced Ca(2+) release' (CICR) mechanism. However, the effects of chronic CaMKII inhibition on Ca(2+)(i) responses during CICR are unknown. We hypothesized that chronic CaMKII inhibition significantly affects CICR in ventricular myocytes. We studied CICR by simultaneously measuring Ca(2+)(i) transients and I(Ca) in voltage-clamped ventricular myocytes isolated from a recently developed genetic mouse model of cardiac CaMKII inhibition. These measurements were repeated in ventricular myocytes from novel mice with cardiac CaMKII inhibition lacking phospholamban (PLN), a known CaMKII substrate and a negative regulator of Ca(2+)(i) uptake into the SR Ca(2+) store. CaMKII inhibition eliminated a pattern of I(Ca) increases called facilitation and significantly reduced beat-to-beat and cell-to-cell variability of peak Ca(2+)(i) transients in ventricular myocytes with PLN. PLN ablation eliminated I(Ca) facilitation even in the absence of CaMKII inhibition and the effects of CaMKII inhibition to reduce SR Ca(2+) content and slow SR Ca(2+) uptake were lost in the absence of PLN. PLN ablation significantly reduced I(Ca) beat-to-beat variability in cells with CaMKII inhibition. These findings show that chronic CaMKII inhibition reduces variability of CICR responses in a manner that is partly dependent on the presence of PLN.     

兔左室三层心肌细胞非特异性阳离子电流的特性

目的研究兔左室除钾电流外是否还存在其它复极外向电流,并进一步研究其在三层心肌细胞上的分布。方法采用胶原酶二步消化法分离兔心肌细胞,用锐利眼科剪分离左室游离壁内、中、外三层心肌,改变灌流液的成份,采用全细胞膜片钳记录离子电流。结果在兔左室肌细胞记录到一种新的电压依赖性、非特异性阳离子电流。该离子通道对Na+、K+、Li+、Cs+通透,对Cl-不通透,能够被Gd3+和La3+阻断。该通道在三层心肌细胞的密度分布不同,中层心肌细胞的电流密度明显小于内层和外层心肌细胞。结论非特异性阳离子电流参与了兔左室心肌细胞的电生理异质性的形成。     

葛根素对大鼠心室肌细胞钠通道的影响

目的研究葛根素对大鼠心室肌细胞膜钠通道电流的影响,探讨葛根素在离子通道水平的作用机制。方法用急性酶解法获得单个大鼠心肌细胞,用标准全细胞膜片钳技术记录钠通道电流。结果0.3,1,3g/L葛根素能剂量依赖性增加钠通道电流,增加值分别(3.4±0.5)%、(34.1±2.7)%和(55.9±2.5)%,n=5。1g/L葛根素能使心肌细胞钠通道电流鄄电压曲线显著下移,最大峰电流密度从(17.4±1.6)pA/pF增加至(23.3±1.8)pA/pF,n=5,P<0.05,但原有的电流鄄电压依赖特征不变;葛根素对钠通道电流失活和复活曲线也无明显影响。1μmol/L普萘洛尔能明显阻断1g/L葛根素增加钠通道电流作用。结论葛根素能浓度依赖性地增加心肌钠大鼠通道电流。     

伊布利特对兔左室中层细胞L型钙电流的影响

目的观察伊布利特对正常心肌细胞L型钙通道电流(ICa-L)的影响。方法用全细胞膜片钳技术记录10-6,10-5mol/L伊布利特细胞外液对兔正常左室中层心肌细胞L型钙通道电流(ICa-L)活性的影响。结果①伊布利特灌流后ICa-LI-V曲线下移,低、高剂量伊布利特灌流后电流密度峰值明显增加(-8.34±2.67,-10.50±3.81pA/pFvs-5.68±1.53pA/pF,P均<0.01),且高剂量较低剂量灌流时增加更明显。②低、高剂量伊布利特灌流后失活曲线右移,且高剂量时右移更明显。高剂量时半数失活电压(V0.5)较低剂量和用药前显著降低,而低剂量与用药前无差异。三种状态的激活曲线无差异。结论伊布利特可能呈浓度依赖性影响心室肌细胞L型钙电流(ICa-L)活性。     

曲美他嗪对缺氧诱导原代大鼠心肌细胞凋亡的影响

目的研究曲美他嗪对缺氧诱导的心肌细胞凋亡及线粒体能量代谢改变的影响。方法采用胰酶和胶原酶联合消化的方法,提取大鼠原代心肌细胞,三气培养箱模拟缺氧损伤。MTT和Hoechst染色检测细胞活性和凋亡,TMRE染色检测线粒体膜电位,Oxygraph-2k细胞呼吸测量仪检测态3、态4呼吸和呼吸控制率,Western blot检测Caspase-3、细胞色素C以及线粒体呼吸链复合酶体Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ蛋白表达水平的变化。结果缺氧能够诱导心肌细胞凋亡、引起线粒体膜电位下降和促进细胞色素C的释放。此外,缺氧能够显著下调态3呼吸和上调态4呼吸,引起呼吸控制率的下降,同时缺氧能够不同程度地下调线粒体呼吸链复合酶体Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ的蛋白表达水平。曲美他嗪能够显著降低缺氧诱导的心肌细胞凋亡、稳定线粒体膜电位和减少细胞色素C释放。此外,曲美他嗪还能减轻缺氧对线粒体呼吸链复合酶体的损伤,维持线粒体有氧呼吸。结论曲美他嗪具有抵抗缺氧致心肌细胞凋亡的作用,可能与其稳定线粒体膜和呼吸链复合酶体有关,继而减少细胞色素C的释放和维持线粒体有氧呼吸。     

The origin of calcium overload in rat cardiac myocytes following metabolic inhibition with 2,4-dinitrophenol

We have investigated the characteristics of the rise in cytoplasmic calcium that occurs when rat isolated cardiac ventricular myocytes are exposed to 2,4-dinitrophenol using conventional and confocal fluorescence microscopy and patch clamp. 2,4-dinitrophenol (200 microM) caused cytoplasmic calcium to increase in two phases: (1) an initial rise in fluo-3 fluorescence of 36+/-2% that was maintained until rigor contraction; (2) a further progressive rise so that fluo-3 fluorescence had increased by 177+/-12% 535 s after 2,4-dinitrophenol addition. Both phases were unaffected by removal of external Ca(2+). 2,4-dinitrophenol caused mitochondrial depolarization, measured using tetramethyl rhodamine ethyl ester fluorescence. Mitochondrial depolarization was associated with a decrease in intra-mitochondrial calcium measured with rhod-2, and experiments on myocytes loaded with both fluo-3 and rhod-2 showed that fluo-3 fluorescence increased as rhod-2 fluorescence fell. The correlation of the onset of the second phase of the increase in cytoplasmic calcium with rigor suggested that this phase was consequent on ATP depletion. DNP also caused activation of an ATP-sensitive potassium current. Depletion of sarcoplasmic reticulum calcium stores by pretreatment with ryanodine, thapsigargin and caffeine prior to the addition of 2,4-dinitrophenol did not affect the initial increase in cytoplasmic calcium, but abolished phase 2. Our results suggest that the initial rise in cytoplasmic calcium seen on application of 2,4-dinitrophenol results from release of mitochondrial calcium because of mitochondrial depolarization, while the second phase is caused by progressive release of calcium from the sarcoplasmic reticulum following depletion of intracellular ATP.     

DNA synthesis in rat atrial myocytes as a response to left ventricular infarction. An autoradiographic study of enzymatically dissociated myocytes

An autoradiographic study was performed on enzymatically isolated atrial muscle cells to examine the DNA synthetic response of atria to left ventricular infarction. DNA synthesis was studied in left and right atrial myocytes and nonmyocytes of: young Sprague-Dawley rats 11 days after ligation of the left coronary artery; rats subjected to a sham surgical procedure without coronary artery ligation; unoperated rats. Each animal received a series of ten injections of tritiated thymidine at 12-h intervals, beginning on the fifth post-operative day; cells were isolated 36 h after the last injection. In infarcted animals, 37.1% of the left atrial myocytes were labeled and binucleated, and 6.5% were labeled and mononucleated; 13% of the right atrial myocytes were labeled and binucleated, while 12.7% were labeled and mononucleated. For both the left and right atria, the incidence of tritiated thymidine label in myocytes of the sham-operated group was similar to that of the unoperated controls, indicating that the surgical procedure did not stimulate DNA synthesis in atrial myocytes. In both left and right atria of the infarcted group, non-muscle cells were labeled to a greater extent (49.9% and 47.1%) than in the sham-operated group (22% and 20.8%), which in turn showed labeling to a greater extent than did the unoperated control group (10.9% and 11.6%), indicating that DNA synthesis was stimulated in non-myocytes of the atria by the sham operation and was further stimulated by experimental infarction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective Vulnerability of Embryonic Cell Populations to Ethanol-Induced Apoptosis: Implications for Alcohol-Related Birth Defects and Neurodevelopmental Disorder

BACKGROUND: Ethanol-induced cell death has been characterized in very few stages of embryogenesis. This investigation comprehensively maps patterns of both programmed and ethanol-induced cell death in the central nervous system and craniofacial region at 0.5-day intervals from gestational day (GD) 6.5 to 11 in mice. METHODS: A teratogenic dosage of ethanol (2.9 g/kg) or vehicle was administered via two intraperitoneal injections to pregnant C57BL/6J mice at various stages of gestation. Cell death patterns were characterized using Nile blue sulfate vital staining and histological analysis of plastic sections. Confocal laser scanning microscopy of LysoTracker Red-stained specimens allowed for three-dimensional visualization of areas of apoptosis and precise sectional imaging of mouse embryos. Apoptosis was also documented using a TUNEL technique on histological sections. RESULTS: Normal programmed cell death in control embryos was noted in the prechordal plate region at GD 8, the neuroepithelium of the fourth ventricle and anterior neuropore at GD 9, and within the ganglia of cranial nerves V, VII-VIII, IX, and X at GD 10. Acute maternal ethanol administration 12 hr before examination resulted in a dramatic increase in apoptosis within sites of programmed cell death in the embryo. Moreover, ethanol-exposed specimens exhibited stage-dependent excessive cell death in other distinct cell populations, particularly within the developing central nervous system. Ethanol-induced apoptosis was notable as follows: GD 7.5-neuroectoderm; GD 8-neural plate and primitive streak; GD 9-alar plate and presumptive neural crest of the rostral hindbrain, especially at the mesencephalon/rhombencephalon junction; GD 9.5-10-branchial arches and rhombomeres; and GD 11-diencephalon, basal ganglia, pons, and developing cerebellum. CONCLUSIONS: The results of this study revealed developmental stage-specific cell populations of the developing brain and craniofacial region that are vulnerable to ethanol-induced apoptosis and provide new insight relative to the genesis of alcohol-related birth defects.     

伊布利特对兔急性心肌梗死后梗死周边区心外膜心室肌细胞L型钙电流的影响

目的观察伊布利特对急性心肌梗死(AMI)后一周心室肌细胞L型钙通道电流(ICa-L)的影响。方法兔开胸,左前降支结扎造成AMI,1周后酶解分离梗死周边区心外膜心室肌细胞,用全细胞膜片钳技术记录10-6mol/L伊布利特细胞外液(伊布利特组)对梗死周边区心外膜心室肌细胞ICa-L活性的影响,并与正常对照组(对照组)及AMI但未灌流伊布利特组(AMI组)比较。结果①AMI 1周时兔梗死周边区心室肌细胞ICa-L受到抑制,电流密度-电压曲线(I-V)上移,ICa-L电流密度峰值降低[-3.52±0.91 pA/pF(n=10)vs-5.68±1.53 pA/pF(n=10),P<0.05];伊布利特组电流密度峰值为-4.84±1.22 pA/pF(n=8),较AMI组显著增大(P<0.05),与对照组比较,虽有减小,但无差异(P>0.05)。②AMI组、伊布利特组ICa-L失活曲线明显左移,以AMI组左移更加明显,对照组半数失活电压(V0.5)为-32±4 mV(n=10),AMI组V0.5增加为-46±7 mV(n=10,P<0.05),伊布利特组V0.5为-36±6mV(n=8),与对照组比较无差异(P>0.05)。结论AMI后1周梗死周边带心室肌细胞L型钙通道受阻滞,伊布利特对缺血引起的ICa-L的异常有明显改善作用。     

乳鼠心室肌起搏电流的特点及其基因的表达

目的研究乳鼠心室肌细胞起搏电流(If)的特点,并运用实时定量聚合酶链式反应(PCR)分析乳鼠心室肌If基因的表达。方法通过酶消化法分离乳鼠心室肌细胞,用光镜、透射电镜、免疫组化鉴定心肌细胞;用全细胞膜片钳技术记录If并研究其特性;采用SYBR-GreenI染料进行实时定量PCR,测定乳鼠心室肌If基因即超极化激活的环核苷酸门控通道(mHCN)亚型2和mHCN4的表达。结果光电显微镜下见搏动频率50~100次/分的细胞团;透射电镜看到丰富的线粒体、肌丝明显;用抗肌动蛋白α-actin的单克隆抗体鉴定心肌细胞,95%以上呈现阳性。全细胞膜片钳记录到心室肌细胞的If,并得到电流密度-电压曲线,其激活电压约为-75mV;实时定量PCR检测mHCN2∶mHCN4为(5.15±0.19)∶1。结论乳鼠心室肌细胞有超极化激活、可被Cs+阻断的If,其基因表达以mHCN2为主。     

Subtype specific roles of beta-adrenergic receptors in apoptosis of adult rat ventricular myocytes

We have previously reported that beta-adrenergic receptor (beta-AR) stimulation promotes apoptosis in adult ventricular myocytes through PKCepsilon-mediated suppression of ERK. In this study, we investigated differential effects of beta-AR subtypes on this signal pathway. The apoptosis induced by the non-specific beta-AR agonist isoproterenol was largely blocked by the beta(1)-selective antagonist CGP 20712A, but not by the beta(2)-selective antagonist ICI 118551. A pro-apoptotic effect of beta(1)-AR was also blocked by the PKA inhibitor H89, while the protein kinase A (PKA) activators forskolin and dibutyryl-cAMP both induced apoptosis. These results indicate that beta(1)-AR-mediated PKA activation is largely responsible for the apoptosis induced by beta-AR in adult rat cardiac myocytes. This conclusion was also supported by the finding that PKA was preferentially activated by beta(1)-AR over beta(2)-AR. beta(2)-AR selectively induced anti-apoptotic ERK activation in the presence of PKCepsilon suppression, and this ERK activation was sensitive to pertussis toxin. PKCepsilon itself as well as Akt, the other anti-apoptotic factor were activated by both beta-AR subtypes. Thus, beta(1)-AR induces pro-apoptotic signals mainly through PKA activation. In contrast, beta(2)-AR is linked to Gi-mediated ERK activation, which is involved in the anti-apoptotic pathway, and is regulated by PKCepsilon. Therefore, our findings suggest a rather complex role for beta-AR subtypes in the regulation of apoptosis in adult ventricular myocytes.     

β2受体阻断药降低梗死后心肌细胞环磷腺苷的含量

目的研究心肌梗死后2-8周心肌细胞β2受体对环磷腺苷(adenosine cyclophosphate,cAMP)含量影响的动态变化,探讨心肌梗死后β2受体在交感神经激动作用中所占地位。方法Wistar大鼠20只随机分为:正常对照组、心肌梗死后2周组、4周组、8周组。分离心肌细胞,每组制备35份样品,按给药不同随机分为7小组,用酶联免疫方法测定心肌细胞环磷腺苷含量。结果在正常和梗死后2、4、8周心肌细胞,β2受体激动可使心肌细胞环磷腺苷含量(pmol／105 cells)分别升高98％、134％、148％和151％(P<0．05)。心肌梗死后,选择性β2受体阻断药对β受体激动引发的心肌细胞环磷腺苷含量的抑制程度增高,选择性β1受体阻断药对其的抑制程度下降,非选择性β受体阻断药对β受体激动引发的正常和心肌梗死后心肌细胞环磷腺苷含量升高均能抑制。结论心肌梗死后β2受体在β受体激动引发的心肌细胞环磷腺苷含量升高作用中所占比例升高。心肌梗死后β2受体在交感神经激动产生的正性肌力和正性变时作用中地位可能提高。     

Cell death signalling mechanisms in heart failure

Cardiac disease is a global epidemic that is on the rise, despite the recent advances in cardiovascular research. Once the myocardium is injured, it has a limited capacity to activate reparative mechanisms to restore proper cardiac function, leading to the development of systemic heart failure. Autophagy, under certain conditions, may result in cell death, further emphasizing the controversial issues regarding the autophagic process as an adaptive or maladaptive biological response. Although significant progress in understanding the signalling mechanisms of cell death in myocytes has been made, the role of apoptotic cell death and programmed necrosis during heart failure is not completely understood. Insight to how myocytes determine whether to activate apoptotic or programmed necrosis signalling machinery remains under current investigation because it is a major problem for both scientists and clinicians in treating heart failure patients. Herein, the different modes of cell death implicated in heart failure are highlighted, as well as the role of B-cell lymphoma-2 family members and how mitochondria act as central organelles in directing such cell death mechanisms.     

Remodelling of Ca handling organelles in adult rat ventricular myocytes during long term culture

It is well known that for cardiomyocytes, isolation and culturing induce largely unknown remodelling processes. We analysed changes in the structure of cell compartments with optical techniques such as confocal microscopy and fluorescence redistribution after photobleaching employing adenoviral-mediated transduction of targeted fluorescent proteins and small molecule dyes. We identified characteristic remodelling processes: the T-tubular membrane system was gradually lost by a process referred to as “sequential pinching off”, in an outward direction. Mitochondria fell in one of three classes, very small (0.9 μm length), medium long (1.8 μm) or extended shape (3.6 μm) organelles. Over the culturing time mitochondria gradually fused. Bleaching of individual mitochondria revealed association between apparently separated mitochondria by “tunnelling” via sub-resolution organelle-tubes. This tunnelling process was increasing over the culturing time. A gradual loss of the cross-striation arrangement in the endoplasmic/sarcoplasmic reticulum was visualised. Analysis of large populations of Ca²⁺ sparks by video-rate confocal 2D-scanning revealed significant albeit small changes of these elementary SR-Ca²⁺ release events in adult cardiomyocytes that could be related to changes in SR-Ca²⁺ content rather than resting Ca²⁺ concentration. In conclusion, primary isolated cardiomyocytes from adult hearts undergo a well-defined, but reproducible subcellular remodelling during optimised long term culture.     

Tyrosine kinases inhibitors block Fas-mediated deleterious effects in normoxic and hypoxic ventricular myocytes

Experimental evidences suggest an important role for Fas receptor activation in a wide range of myocardial pathologies, which are associated with a variety of arrhythmias and mechanical dysfunction. Our recent studies have shown that Fas-mediated arrhythmias and mechanical disturbances of ventricular myocytes can be accounted for by activation of the phospholipase C-1,4,5-inositol triphosphate-intracellular calcium release (PLC-1,4,5-IP(3)-[Ca(2+)](i)) cascade, which is responsible for attenuating the transient outward current (I(to)) and augmenting the L-type Ca(2+) current (I(Ca,L)). We have also shown that whereas ventricular myocytes are resistant to Fas-mediated apoptosis, hypoxia predisposes myocytes to apoptosis induced by Fas activation by shifting the balance between pro-apoptotic and anti-apoptotic proteins towards the former. Since protein tyrosine phosphorylation has been shown to be involved in Fas signaling, we have hypothesized that inhibiting tyrosine kinases will attenuate Fas-mediated effects in ventricular myocytes in normoxic and hypoxic conditions. Therefore, we tested the effect of the tyrosine kinases inhibitors, genistein (50 micromol/l) and herbimycin A (50 microg/ml), on the abovementioned Fas-mediated effects in cultured neonatal rat ventricular myocytes (NRVM) and in freshly isolated adult murine ventricular myocytes. Fas receptor was activated by incubating NRVM with recombinant Fas ligand (rFasL, 50 ng/ml) and enhancing antibody (1 microg/ml), or by incubation of murine ventricular myocytes with the Fas-activating antibody Jo2 (10 microg/ml). The major findings were that genistein prevented Fas-mediated increase in 1,4,5-IP(3) production in NRVM (quantified by ion-exchange chromatography): 216 +/- 41 counts per minute (cpm) in control, 605 +/- 184 cpm in FasL-treated cardiomyocytes and 137 +/- 51 cpm in rFasL + genistein-treated cultures. Accordingly, genistein or herbimycin A abolished the diastolic [Ca(2+)](i)-rise (as measured by fura-2 fluorescence) and arrhythmogenic activity in both rat and murine ventricular myocytes, and the Fas-mediated changes in I(to) and I(Ca,L) in murine ventricular myocytes. Importantly, genistein attenuated Fas-mediated apoptosis in hypoxic (22 h in 1% O(2)) NRVM; the apoptotic ratios as measured by DAPI fluorescence assay were: 4.6 +/- 1.0% in control, 12.5 +/- 3.0% in rFasL and 7.3 +/- 1.6% in rFasL + genistein-treated NRVM. This prevention of apoptosis by tyrosine kinases blockade was accompanied by inhibition of hypoxia-induced increased Fas expression and decreased expression of the anti-apoptotic protein xIAP. In conclusion, our findings indicate that tyrosine phosphorylation is involved in Fas signaling in ventricular myocytes, and can, therefore, serve as a novel potential target for attenuating Fas-mediated dysfunction in normoxic and hypoxic myocardium.     

Acute vincristine pretreatment protects adult mouse cardiac myocytes from oxidative stress

Vincristine is a chemotherapeutic agent that disrupts microtubules. We noted that paclitaxel (Taxol), which stabilizes microtubules, protected cultured adult mouse cardiac myocytes from oxidative stress induced by H(2)O(2). We hypothesized that vincristine, which disrupts microtubules, should have the opposite effect. To our surprise, we found that pretreatment with concentrations of vincristine ranging from 30 to 120 micromol/L for 60 min preserved myocyte viability and morphology after incubation with 30 micromol/L of H(2)O(2) for 35 min as measured by trypan blue exclusion. The cardioprotective effects of vincristine were also observed during prolonged hypoxia. With continuous exposure to vincristine, survival lasted for as long as 24 h, but longer periods of exposure up to 42 h resulted in extensive cell death. Despite microtubule disruption evidenced on deconvolution microscopy, vincristine activated a prosurvival pathway resulting in increased phosphorylation of Akt, ERK and GSK-3beta and in reduced cytochrome C release into the cytosol. Pharmacological inhibitors of Akt and Erk attenuated the cardioprotective effect of vincristine. We conclude that short-term pretreatment with vincristine exerts dramatic protective effects in cultured adult mouse myocytes subjected to acute oxidative stress. Despite causing microtubule disruption, vincristine initiates a prosurvival signaling pathway. As vincristine and doxorubicin are often used in conjunction to treat patients, it is possible that vincristine could be used to modify the cardiotoxicity of doxorubicin.