TNF‐α‐induced cardiomyocyte apoptosis contributes to cardiac dysfunction after coronary microembolization in mini‐pigs

This experimental study was designed to clarify the relationship between cardiomyocyte apoptosis and tumour necrosis factor‐alpha (TNF‐α) expression, and confirm the effect of TNF‐α on cardiac dysfunction after coronary microembolization (CME) in mini‐pigs. Nineteen mini‐pigs were divided into three groups: sham‐operation group (n = 5), CME group (n = 7) and adalimumab pre‐treatment group (n = 7; TNF‐α antibody, 2 mg/kg intracoronary injection before CME). Magnetic resonance imaging (3.0‐T) was performed at baseline, 6th hour and 1 week after procedure. Cardiomyocyte apoptosis was detected by cardiac‐TUNEL staining, and caspase‐3 and caspase‐8 were detected by RT‐PCR and immunohistochemistry. Furthermore, serum TNF‐α, IL‐6 and troponin T were analysed, while myocardial expressions of TNF‐α and IL‐6 were detected. Both TNF‐α expression (serum level and myocardial expression) and average number of apoptotic cardiomyocyte nuclei were significantly increased in CME group compared with the sham‐operation group. Six hours after CME, left ventricular end‐systolic volume (LVESV) was increased and the left ventricular ejection fraction (LVEF) was decreased in CME group. Pre‐treatment with adalimumab not only significantly improved LVEF after CME (6th hour: 54.9 ± 2.3% versus 50.4 ± 3.9%, P = 0.036; 1 week: 56.7 ± 4.2% versus 52.7 ± 2.9%, P = 0.041), but also suppressed cardiomyocyte apoptosis and the expression of caspase‐3 and caspase‐8. Meanwhile, the average number of apoptotic cardiomyocytes nuclei was inversely correlated with LVEF (r = ?0.535, P = 0.022). TNF‐α‐induced cardiomyocyte apoptosis is likely involved in cardiac dysfunction after CME. TNF‐α antibody therapy suppresses cardiomyocyte apoptosis and improves early cardiac function after CME.     

Tumor necrosis factor-alpha in mechanic trauma plasma mediates cardiomyocyte apoptosis

Mechanical traumatic injury causes cardiomyocyte apoptosis and cardiac dysfunction. However, the signaling mechanisms leading to posttraumatic cardiomyocyte apoptosis remains unclear. The present study attempted to identify the molecular mechanisms responsible for cardiomyocyte apoptosis induced by trauma. Normal cardiomyocytes (NC) or traumatic cardiomyocytes (TC; isolated immediately after trauma) were cultured with normal plasma (NP) or traumatic plasma (TP; isolated 1.5 h after trauma) for 12 h, and apoptosis was determined by caspase-3 activation. Exposure of TC to NP failed to induce significant cardiomyocyte apoptosis. In contrast, exposure of NC to TP resulted in a greater than twofold increase in caspase-3 activation (P < 0.01). Incubation of cardiomyocytes with cytomix (a mixture of TNF-alpha, IL-1beta, and IFN-gamma) or TNF-alpha alone, but not with IL-1beta or IFN-gamma alone, caused significant caspase-3 activation (P < 0.01). TP-induced caspase-3 activation was virtually abolished by an anti-TNF-alpha antibody, and TP isolated from TNF-alpha(-/-) mice failed to induce caspase-3 activation. Moreover, incubation of cardiomyocytes with TP upregulated inducible nitric oxide (NO) synthase (iNOS)/NADPH oxidase expression, increased NO/superoxide production, and increased cardiomyocyte protein nitration (measured by nitrotyrosine content). These oxidative/nitrative stresses and the resultant cardiomyocyte caspase-3 activation can be blocked by neutralization of TNF-alpha (anti-TNF-alpha antibody), inhibition of iNOS (1400W), or NADPH oxidase (apocynin) and scavenging of peroxynitrite (FP15) (P < 0.01). Taken together, our study demonstrated that there exists a TNF-alpha-initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to posttraumatic myocardial apoptosis. Therapeutic interventions that block this signaling cascade may attenuate posttraumatic cardiac injury and reduce the incidence of secondary organ dysfunction after trauma.     

TNF-alpha-mediated cardiomyocyte apoptosis involves caspase-12 and calpain

Following ischemia-reperfusion, there is a sustained increase of TNF-alpha both locally in the heart as well as in circulating levels in blood. While TNF-alpha has been implicated in cardiomyocyte apoptosis which occurs in several cardiomyopathies, the molecular pathways by which TNF-alpha induces apoptosis in these cells are not fully elucidated. We investigated the role of the two families of cysteine proteases, caspases and calpains, which are known to participate in apoptotic cell death. The effect of the highly specific calpain inhibitor, Z-LLY-fmk, and the caspase pathways involved in TNF-alpha-mediated apoptosis of the HL-1 cardiomyocyte cell line were examined. Activation of the downstream caspase-3, and the cleavage of poly ADP-ribose polymerase (PARP) were observed in a time-dependent manner upon treatment with TNF-alpha. Caspase-12, but not caspase-9, was activated in response to TNF-stimulation, indicating that an endoplasmic reticulum (ER)/calcium-dependent pathway may be involved. In HL-1 cardiomyocytes, TNF-alpha-induced apoptosis appears to be mediated by calpain as apoptotic changes were abrogated in the presence of the highly specific calpain inhibitor, Z-LLY-fmk. In conclusion, our results suggest that TNF-alpha-mediated apoptosis in HL-1 cardiomyocytes follows the caspase-12 apoptotic pathway that involves calpain.     

Involvement of death receptor signaling in mechanical stretch-induced cardiomyocyte apoptosis

Recent evidences suggest that mechanical overload associated with abnormal blood pressure causes apoptosis in cardiovascular system. Still, the intracellular signaling leading to cardiomyocyte apoptosis has not been fully defined. Previous reports ascribed stretch-induced cardiomyocyte apoptosis to rennin-angiotensin-system (RAS) signaling and/or mitochondria-dependent apoptosis pathway. The present study shows the involvement of death receptor signaling in mechanical stretch-induced cardiomyocyte apoptosis. By employing a well-described in vitro stretch model, we studied stretch-induced apoptosis and found that the death receptor-mediated apoptotic signaling was activated in stretch-induced apoptosis in neonatal rat cardiomyocytes. The major finding are as following: (1) The mechanical stretch activated death receptor-mediated apoptotic signaling in cardiomyocytes, including activation of caspases 8, 9 and 3, up-regulation of Fas, FasL expression and cell surface trafficking of death ligands (FasL and TRAIL); (2) That exogenous death ligand (TRAIL) enhanced, while soluble death receptor (sDR5) neutralized, stretch-induced apoptosis; (3) Adenovirus-delivered dominant negative FADD (FADD-DN) significantly reduced apoptosis, caspases 8, 9, and 3 activation, and stretch-induced cyt c release from mitochondria. These data clearly suggested mechanical stretch activated death receptor-mediated apoptotic signaling in cardiomyocytes. In conclusion, our data suggest that the FADD-linked death receptor signaling may contribute to stretch-induced cardiomyocyte apoptosis, probably through activating mitochondria-dependent apoptotic signaling.     

Reactive oxygen species induce cardiomyocyte apoptosis partly through TNF-alpha

Many studies have indicated that oxidative stress induces apoptosis in cardiomyocytes, but its mechanism remains unknown. We examined whether tumor necrosis factor-alpha (TNF-alpha) is involved in oxidative stress-induced cardiomyocyte apoptosis. Pretreatment with anti-TNF-alpha antibody significantly decreased the number of H(2)O(2)-induced TUNEL-positive cardiomyocytes. Expression of TNF-alpha gene was upregulated by H(2)O(2), and H(2)O(2) mildly but significantly increased the concentration of TNF-alpha in the culture medium. Although neither low dose of H(2)O(2) nor TNF-alpha induced apoptosis, stimulation with H(2)O(2) and TNF-alpha synergistically increased apoptosis. These results suggest that oxidative stress induces apoptosis of cardiac myocytes partly through TNF-alpha.     

Focal adhesion kinase as a RhoA-activable signaling scaffold mediating Akt activation and cardiomyocyte protection

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) J. Biol. Chem. 282, 8069-8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with sphingosine 1-phosphate recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF271 and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.     

Regulation of cardiomyocyte apoptosis in ischemic reperfused mouse heart by glutathione peroxidase

Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this is also true for other animals, an isolated perfused mouse heart was subjected to 30 min of ischemia followed by 2 h of reperfusion. Experiments were terminated before ischemia (baseline), after ischemia, and at 30, 60, 90 and 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The results of our study revealed that apoptotic cells appear only after 60 min of reperfusion as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). Since our previous studies showed a role of glutathione peroxidase (GSHPx) in apoptotic cell death, we performed identical experiments using isolated hearts from GSHPx-l knockout mice and transgenic mice overexpressing GSHPx-l. GSHPx-l knockout mice showed evidence of apoptotic cell death even after 30 min of reperfusion. Significant number of apoptotic cells were found in the cardiomyocytes as compared to non-transgenic control animals. To the contrary, very few apoptotic cells were found in the hearts of the transgenic mice overexpressing GSHPx-l. Hearts of GSHPx-l knockout mice were more susceptible to ischemia/reperfusion injury while transgenic mice overexpressing GSHPx- 1 were less susceptible to ischemia reperfusion injury compared to non-transgenic control animals. The results of this study clearly demonstrate a role of GSHPx in ischemia/reperfusion-induced apoptosis in mouse heart.     

Postconditioning attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways

A sequence of intermittent interruptions of oxygen supply (i.e., postconditioning, Postcon) at reoxygenation reduces oxidant-induced cardiomyocyte loss. This study tested the hypothesis that prevention of cardiomyocyte apoptosis by Postcon is mediated by mitogen-activated protein kinases pathways. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Relative to hypoxia alone, reoxygenation stimulated expression of JNKs and p38 kinases, corresponding to increased activity of JNKs (phospho-c-Jun) and p38 (phospho-ATF2). The level of TNFα in cell lysates, activity of cytosolic caspases-8, -3, expression of Bax and the number of apoptotic cardiomyocytes were increased while expression of Bcl-2 was decreased with reoxygenation. Consistent with an attenuation in generation of superoxide anions detected by lucigenin-enhanced chemiluminescence at early period of reoxygenation, treatment of cardiomyocytes with Postcon further reduced expression and activity of JNKs and p38 kinases, level of TNFα, the frequency of apoptotic cells and expression of Bax. However, the inhibitory effects of Postcon on these changes were lost when its application was delayed by 5 min after the start of reoxygenation. Addition of a JNK/p38 stimulator, anisomycin into cardiomyocytes at the beginning of reoxygenation eliminated protection by Postcon. These data suggest that 1) hypoxia/reoxygenation elicits cardiomyocyte apoptosis in conjunction with expression and activation of JNK and p38 kinases, release of TNFα, activation of caspases, and an increase in imbalance of pro-/anti-apoptotic proteins; 2) Postcon attenuates cardiomyocyte apoptosis, potentially mediated by inhibiting JNKs/p-38 signaling pathways and reducing TNFα release and caspase expression.     

Human intestinal fibroblasts prevent apoptosis in human intestinal mast cells by a mechanism independent of stem cell factor, IL-3, IL-4, and nerve growth factor

In rodents, fibroblasts (FBs) mediate stem cell factor (SCF)-dependent growth of mast cells (MCs). In humans, SCF is mandatory for MC differentiation and survival. Other factors such as IL-3, IL-4, and nerve growth factor (NGF) act in synergism with SCF, thus enhancing proliferation and/or preventing apoptosis in MCs. In this study, we studied in vitro interactions between human MCs and human FBs, both isolated from the intestine and purified to homogeneity. In coculture with FBs, MCs survived for up to 3 wk, whereas purified MCs cultured alone died within a few days. TNF-alpha and IL-1beta, which both did not affect MC survival directly, enhanced FB-dependent MC growth. We provide evidence that FB-derived MC growth factors are soluble, heat-sensitive molecules which down-regulate MC apoptosis without enhancing MC proliferation. However, only low amounts of SCF were measured in FB-conditioned medium (<0.2 ng/ml). Moreover, blocking of SCF/c-kit interaction by anti-SCF or anti-c-kit Abs and neutralization of IL-3, IL-4, and NGF did not affect MC survival in the coculture system. In conclusion, our data indicate that human FBs promote survival of human MCs by mechanisms independent of SCF, IL-3, IL-4, and NGF. Such interactions between MCs and FBs may explain why MCs accumulate at sites of inflammatory bowel disease and intestinal fibrosis.     

Chloride channel inhibition prevents ROS-dependent apoptosis induced by ischemia-reperfusion in mouse cardiomyocytes.

Apoptosis of cardiomyocytes following ischemia and Apoptosis of cardiomyocytes following ischemia and known about the mechanism by which it is induced. Recently, essential roles of a Cl- channel whose activity triggers the apoptotic volume decrease and of reactive oxygen species (ROS) in activation of this channel have been identified in mitochondrion-mediated apoptosis. Therefore, in this study, involvement of Cl- channels and ROS in apoptosis was studied in primary mouse cardiomyocyte cultures subjected to ischemia-reperfusion. Apoptotic cell death as measured by caspase-3 activation, chromatin condensation, DNA laddering, and cell viability reduction was observed tens of hours after reperfusion but never immediately after ischemia. A non-selective Cl-channel blocker (DIDS or NPPB) rescued cells from apoptotic death when applied during the reperfusion, but not ischemia, period. Another blocker relatively specific to the volume-sensitive outwardly rectifying (VSOR) Cl-channel (phloretin) was also effective in protecting ischemic cardiomyocytes from apoptosis induced by reperfusion. A profound increase in intracellular ROS was detected in cardiomyocytes during the reperfusion, but not ischemia, period. Scavengers for ROS, H2O2 and superoxide all inhibited apoptosis induced by ischemia-reperfusion. Thus, it is concluded that the mechanism by which cardiomyocyte apoptosis is induced by ischemia-reperfusion involves VSOR Cl- channel activity and intracellular ROS production.     

Downregulation of endothelin-1 by interleukin-4 during gastric ulcer healing.

We investigated the course of events associated with gastric ulcer healing by analyzing mucosal expression of interleukin-4 (IL-4), endothelin-1 (ET-1), tumor necrosis factor-alpha (TNF-alpha), and the activity of constitutive (cNOS) and inducible nitric oxide synthase (NOS-2). Ulcer onset was characterized by a massive epithelial apoptosis associated with a 5.7-fold increase in TNF-alpha, a 17.5-fold increase in NOS-2, and a 3.9-fold increase in ET-1, while mucosal expression of cNOS showed a 7.6-fold drop and IL-4 fell by 37.2%. Healing was accompanied by a rapid raise in IL-4; decrease in apoptosis, TNF-alpha, ET-1, and NOS-2; and a slow recovery in cNOS. The expression of IL-4 returned to control levels by the 7th day of healing and that of ET-1 and TNF-alpha by the 14th day, while apoptotic DNA fragmentation and the activity of NOS-2 remained significantly elevated beyond the 14-day period. The results suggest that a decrease in the mucosal level of IL-4 at ulcer onset may well be a key factor causing dysregulation of ET-1 production, induction of TNF-alpha, and triggering the apoptotic events that affect the efficiency of mucosal repair.     

Role of interleukin-6 in cardiac inflammation and dysfunction after burn complicated by sepsis

To examine the role of myocardial interleukin-6 (IL-6) in myocardial inflammation and dysfunction after burn complicated by sepsis, we performed 40% total body surface area contact burn followed by late (7 days) Streptococcus pneumoniae pneumonia sepsis in wild-type (WT) mice, IL-6 knockout (IL-6 KO) mice, and transgenic mice overexpressing IL-6 in the myocardium (TG). Twenty-four hours after sepsis was induced, isolated cardiomyocytes were harvested and cultured in vitro, and supernatant concentrations of IL-6 and tumor necrosis factor (TNF)-alpha were measured. Cardiomyocyte intracellular calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) concentrations were also determined. Separate mice in each group underwent in vivo global hemodynamic and cardiac function assessment by cannulation of the carotid artery and insertion of a left ventricular pressure volume conductance catheter. Hearts from these mice were collected for histopathological assessment of inflammatory response, fibrosis, and apoptosis. In the WT group, there was an increase in cardiomyocyte TNF-alpha, [Ca(2+)](i), and [Na(+)](i) after burn plus sepsis, along with cardiac contractile dysfunction, inflammation, and apoptosis. These changes were attenuated in the IL-6 KO group but accentuated in the TG group. We conclude myocardial IL-6 mediates cardiac inflammation and contractile dysfunction after burn plus sepsis.     

缺氧诱导心肌细胞凋亡与Caspase-3激活及细胞内钙超载的关系

目的: 研究缺氧对心肌细胞Caspases的激活效应与细胞内钙的关系.方法: 将培养的心肌细胞暴露于95% N2/5% CO2混合气体培养室进行缺氧处理,缺氧0、30 min、1、3、6、12、24 h后,应用激光共聚焦显微镜检测缺氧早期心肌细胞胞浆游离钙的变化;应用细胞内钙螯合剂、Caspase-1与Caspase-3特异性抑制剂预处理心肌细胞后缺氧12 h,检测DNA片段化与Caspase-3活性;Western blot、RT-PCR方法分别检测细胞线粒体与胞浆中Cyt c蛋白的变化以及Caspase-3 mRNA表达的改变.结果: 缺氧后30 min心肌细胞胞浆游离钙即显著增高,1 h达到峰值.缺氧3 h线粒体Cyt c无显著变化,而胞浆中Cyt c开始增多,至缺氧12 h,线粒体中Cyt c仅见一弱阳性条带,而胞浆呈强阳性反应,缺氧24 h 线粒体中已不能检测到Cyt c.缺氧3 h心肌细胞Caspase-3 mRNA上调,在观察的24 h内持续处于高表达状态.分别应用Caspase-3抑制剂和细胞内钙螯合剂预处理心肌细胞后均可使心肌细胞Caspase-3活性降低并完全阻断DNA片段化发生,细胞内钙螯合剂与Caspase-3抑制剂联合应用使凋亡细胞百分率降低的程度比分别单独使用两者更明显.结论: 缺氧可致心肌细胞线粒体Cyt c释放至胞浆,导致Caspase途径激活,从而导致细胞凋亡.缺氧所致心肌细胞钙超载在时序上早于线粒体Cyt c释放与Caspase-3的激活,螯合细胞内钙可阻断缺氧诱导的Caspase-3激活,并拮抗心肌细胞凋亡的发生,因此细胞钙超载是启动线粒体Cyt c释放与Caspase-3激活的一个早期重要分子事件.     

乙醛脱氢酶2可减少氧化损伤诱导的心肌细胞凋亡

乙醛脱氢酶2 (aldehyde dehydrogenase 2, ALDH2)是线粒体特异性酶,已被证明参与氧化应激诱导的细胞凋亡,而在心肌细胞中的作用知之甚少。本研究旨在通过用特异性ALDH2抑制剂大豆苷抑制ALDH2活性来研究ALDH2在抗霉素A诱导的心肌细胞凋亡中的作用。应用抗霉素A和大豆苷诱导小鼠心肌细胞,然后测定ALDH2酶活性、细胞内活性氧(reactive oxy gen species, ROS)含量和细胞凋亡,应用RT-PCR和蛋白质印迹法(Western blotting)检测ALDH2 m RNA和蛋白表达。结果表明,抗霉素A (40μg/mL)可诱导新生心肌细胞凋亡,而大豆苷(50μmol/L)能有效地抑制ALDH2活性而对细胞凋亡没有影响,并且可显著增强抗霉素A诱导的心肌细胞凋亡(53.72%～71.33%, p<0.05)。与单独用抗霉素A处理的细胞相比,抗霉素A和大豆苷共处理的心肌细胞中活化的丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号传导途径(p38-MAPK)的磷酸化也显著增加。本研究初步表明,改变线粒体ALDH2活性可能是减少氧化损伤诱导的心肌细胞凋亡的潜在选择。     

Electronegative low-density lipoprotein induces cardiomyocyte apoptosis indirectly through endothelial cell-released chemokines

Cardiomyocyte apoptosis has a critical role in the pathogenesis of heart failure. L5, the most negatively charged subfraction of human plasma low-density lipoprotein (LDL), induces several atherogenic responses in endothelial cells (ECs), including apoptosis. We hypothesized that L5 also contributes to cardiomyocyte apoptosis and studied whether it does so indirectly by inducing the secretion of factors from ECs. We examined apoptosis of rat cardiomyocytes treated with culture-conditioned medium (CCM) of rat ECs that were exposed to L5 or L1 (the least negatively charged LDL subfraction). Apoptosis at early and late time points was twofold greater in cardiomyocytes treated with L5 CCM than in those treated with L1 CCM. The indirect effect of L5 on cardiomyocyte apoptosis was significantly reduced by pretreating ECs with inhibitors of phosphatidylinositol 3-kinase (PI3K) or CXC receptor 2 (CXCR2). Studies with cytokine protein arrays revealed that L5 CCM, but not L1 CCM, contained high levels of ELR(+) CXC chemokines, including lipopolysaccharide-induced chemokine (LIX) and interleukin (IL)-8. The L5-induced release of these chemokines from ECs was abolished by inhibiting the lectin-like oxidized LDL receptor-1 (LOX-1). Addition of recombinant LIX or IL-8 to CCM-free cardiomyocyte cultures increased apoptosis and enhanced production of tumor necrosis factor (TNF)-α and IL-1β by increasing the translocation of NF-κB into the nucleus; these effects were attenuated by inhibiting PI3K and CXCR2. In conclusion, L5 may indirectly induce cardiomyocyte apoptosis by enhancing secretion of ELR(+) CXC chemokines from ECs, which in turn activate CXCR2/PI3K/NF-κB signaling to increase the release of TNF-α and IL-1β.     

CircITGA7对缺血性心律失常大鼠心肌细胞凋亡的影响

研究环状RNA ITGA7(circITGA7)在心律失常大鼠心肌细胞中的表达,并探讨其对心律失常大鼠心肌细胞凋亡的影响及机制。40只SPF级SD雄性大鼠,分为4组,分别为假手术组、心律失常组、circITGA7干扰心律失常组和对照干扰心律失常组,尾静脉注射circITGA7干扰腺相关病毒构建circITGA7干扰大鼠模型,并通过手术结扎冠状动脉前降支建立缺血性心律失常大鼠模型。通过荧光定量PCR法(Q-PCR)检测circITGA7在心律失常大鼠心肌组织中的表达水平。分别检测circITGA7对心律失常大鼠心肌氧化应激和凋亡的影响;Western blotting检测circITGA7对心律失常大鼠心肌细胞p-AKT和ITGA7蛋白表达的影响。Q-PCR结果显示circITGA7在心律失常大鼠心肌组织中的表达显着高于假手术组大鼠;与对照干扰心律失常组比较,circITGA7干扰心律失常组大鼠心律失常评分显著降低,差异具有统计学意义(p<0.01)。与假手术组比较,心律失常组大鼠心肌细胞SOD活力显著降低,MDA含量显著升高;与对照干扰心律失常组比较,circITGA7干扰心律失常组大鼠心律失常组大鼠心肌细胞SOD活力显著升高,MDA含量显著降低,差异具有统计学意义(p<0.01)。TUNEL检测结果显示,与假手术组比较,心律失常大鼠心肌细胞凋亡程度显著升高;与对照干扰心律失常组大鼠比较,circITGA7干扰心律失常组大鼠心肌细胞凋亡率为显著降低(p<0.01);Western blotting结果显示,与假手术组比较,心律失常大鼠心肌细胞p-AKT和ITGA7的蛋白表达水平均显著升高;与对照干扰心律失常组比较,circITGA7干扰心律失常组大鼠心肌细胞p-AKT和ITGA7的蛋白表达水平均显著下降(p<0.01)。干扰circITGA7能够抑制AKT路径,抑制心律失常大鼠心肌细胞氧化应激和细胞凋亡,缓解大鼠心律失常。     

Akt regulates IL-10 mediated suppression of TNFα-induced cardiomyocyte apoptosis by upregulating Stat3 phosphorylation

Background

We have already reported that TNF-α increases cardiomyocyte apoptosis and IL-10 treatment prevented these effects of TNF-α. Present study investigates the role of Akt and Jak/Stat pathway in the IL-10 modulation of TNF-α induced cardiomyocyte apoptosis.

Methodology/Principal findings

Cardiomyocytes isolated from adult Sprague Dawley rats were exposed to TNF-α (10 ng/ml), IL-10 (10 ng/ml) and TNF-α+IL-10 (ratio 1) for 4 h. Exposure to TNF-α resulted in an increase in cardiomyocyte apoptosis as measured by flow cytometry and TUNEL assay. IL-10 by itself had no effect, but it prevented TNF-α induced apoptosis. IL-10 treatment increased Akt levels within cardiomyocytes and this change was associated with an increase in Jak1 and Stat3 phosphorylation. Pre-exposure of cells to Akt inhibitor prevented IL-10 induced Stat3 phosphorylation. Furthermore, in the presence of Akt or Stat3 inhibitor, IL-10 treatment was unable to block TNF-α induced cardiomyocyte apoptosis.

Conclusion

It is suggested that IL-10 modulation of TNF-α induced cardiomyocyte apoptosis is mediated by Akt via Stat3 activation.     

Inhibition of phosphatidylinositol-3-kinase blocks development of functional embryonic cardiomyocytes

Culturing murine embryonic stem (ES) cells within embryoid bodies (EBs) has been reported to reproduce cardiomyocyte development from primitive precursor cells to highly specialized phenotypes of cardiac tissue. We show here that the specific inhibitor of phosphatidylinositol-3-kinase (PI-3-kinase), LY294002, blocks the growth and induces apoptosis as well as necrosis of D3 ES cells within early EBs. Treatment of EBs from day 3 to day 7 with 50 microM LY294002 resulted in a massive loss of alpha-actinin-stained cardiomyocytes after plating the EBs for additional 7 days. In parallel we observed a strong decrease in the number of EBs containing area(s) with beating cardiomyocytes. The specific action of the PI-3-kinase inhibitor on development of cardiomyocytes was demonstrated by the observation that formation of endothelial cells was not affected in the same EBs. Our results provide the first evidence that signal transduction via the PI-3-kinase pathway is essential for mammalian early cardiomyocyte development.