缺血再灌注所致心肌细胞凋亡的分子机制——Homer1a和QKI的作用
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摘要
心肌梗死是人类的头号杀手,目前通过溶栓或者经皮冠状动脉支架置入术(PCI)等方法快速有效的恢复冠脉血流是减轻心肌损伤的最佳策略。然而,这种心肌再灌注自身亦可以引起心肌损伤,可能导致了最终梗死面积的50 %,这种损伤被称为心肌再灌注损伤。心肌再灌注损伤是一种多种分子和信号转导通路参与,损伤机制与保护机制相互竞争的复杂过程。因此,找到导致或者抑制心肌缺血/再灌注损伤的重要分子能够帮助我们在基础研究或者临床应用中最大程度的利用心肌再灌注的有益作用。本研究选择两个在心肌中也大量表达,但在心肌中功能尚不清楚的两个分子(Homer 1a蛋白和QKI蛋白),试图探明其在心肌再灌注损伤中的表达变化及意义。
研究一、Homer 1脚架蛋白Homer在脑、心脏和骨骼肌,肾脏等组织表达。已有研究主要集中于神经系统,所有Homer蛋白都通过其N -末端EVH1域与其他含有脯氨酸富集的基序(PPxxFr)的分子相互作用。所有长Homer亚型可以通过C -末端循环域含有亮氨酸拉链基序与同族或其他Homer家庭成员形成同源、异源二聚体。由于其独特的二聚性能,长型的Homer作为支架蛋白调节多种信号通路;与此相反,短型的Homer,如Homer1a,缺乏二聚体形成结构域,常抑制长型行使的功能。神经系统的研究表明,ERK的激活依赖于Homer 1b/c,而Homer1a可以干扰ERK激活。ERK在心肌中可以抑制心肌细胞凋亡,在再灌注过程中发挥一种心肌保护效应。本研究试图观察Homer 1各亚型在心肌缺血/再灌注中的表达模式,探明其对缺血再灌注所致心肌细胞凋亡的影响及其可能的机制。主要结果如下:
1.离体和在体实验都证明缺血/再灌注可以诱导Homer1a上调
我们采用商业抗体检测了新生大鼠心肌细胞、H9C2细胞中Homer 1各亚型的表达情况。神经系统中,Homer 1a只有在神经元活动或者受到刺激时才表达。而在我们的观察中,正常心肌细胞可以表达Homer 1a和1b/c,与神经系统略有不同。在培养的新生大鼠心肌细胞和胚心来源的成肌细胞H9C2细胞中采用培养基和氧气同时撤除的方式模拟细胞缺血,Homer 1a都被缺血/再灌注大量诱导表达。与Homer 1a不同,Homer 1b/c在以上处理中均保持稳定。
2.沉默Homer 1a增加H9C2的凋亡敏感性
为了评价Homer 1a在缺血/再灌注条件下上调的意义,我们设计了针对Homer 1a的siRNA以敲除Homer 1a的表达。敲除之后的细胞再给予9 hr缺血和6 hr再灌注处理,采用流式细胞仪检测细胞凋亡率。转染了阴性对照RNA的细胞凋亡率为43.6±3.4 %,而转染了siRNA的细胞凋亡率为21.2±3.3 %,显著低于ncRNA组(P<0.05)。这些数据提示IR时Homer 1a的上调促进了心肌细胞凋亡。
3.过表达Homer 1a增加乳鼠心肌细胞凋亡易感性
为了确认Homer 1a是否促进心肌细胞凋亡,我们构建了表达Homer 1a的腺病毒。采用对照病毒(Ad-RFP)或者Homer 1a病毒(Ad-H1a)感染心肌细胞,然后再给予4 hr缺血和6 hr再灌注处理,采用Annexin V单染,流式细胞仪检测凋亡率,未经处理的细胞由于对胰酶的敏感,也产生了3.8±0.8 %的凋亡;感染Ad-RFP的细胞受到上述I4R6处理之后凋亡率为31.7±4.5 %;而感染Ad-Homer的细胞经I4R6处理之后凋亡率为52.8±2.3 %,显著高于Ad-RFP组。
4.沉默Homer 1a增加H9C2细胞中IR诱导的ERK1/2激活
ERK1/2是一种具有心肌保护作用的激酶,过表达组成性活化的MEK1/2(ERK1/2上游激酶)可以缩小IR引起的心肌梗死范围。沉默Homer 1a之后,4 hr缺血加6小时再灌注都不能诱导Homer 1a增高。4 hr缺血和20 min再灌注之后,ERK1/2被激活,而在Homer 1a沉默的细胞中,ERK1/2激活的程度更加显著。
5.过表达Homer 1a抑制IR过程中ERK1/2的激活
为了检验Homer 1a对心肌中ERK1/2的激活是否有抑制作用,腺病毒感染乳鼠心肌细胞后给予4 hr缺血加20 min再灌注。我们发现,IR在对照病毒感染的细胞中诱导了ERK1/2的显著激活;而采用腺病毒感染过表达Homer 1a不仅可以抑制ERK1/2的本底磷酸化水平,还可以抑制IR诱导的ERK1/2激活。
总之,以上研究首次发现IR可以诱导Homer 1a表达上调,这种上调促进心肌细胞凋亡,可能的机制为Homer 1a的上调抑制了心肌保护激酶ERK1/2的充分激活。
研究二、QKI蛋白QKI属于信号转导和RNA激活(STAR)家族蛋白,包括QKI-5、-6、-7三种异构体,qkI的初始转录本C末端外显子选择性剪接后表达。每一种QKI异构体都含有KH RNA结合结构域。QKI蛋白在脑和心脏大量表达并通过调节mRNA代谢发挥作用。QKI在神经系统中的作用得到了广泛的研究,证明其可以通过调控参与髓鞘生成过程中相关mRNA的稳态而参与髓鞘生成,QKI缺乏可以导致髓鞘生成障碍。由于QKI蛋白通过特异结合其靶mRNA的3’UTR起作用,通过生物信息学分析,有很多mRNA可能是QKI蛋白的靶分子,其中促凋亡蛋白Foxo 1 mRNA的3’UTR有3个QKI保守结合位点。因此我们致力于研究QKI蛋白在心肌细胞凋亡中的作用,并试图探明其与FOXO1之间的调控关系。主要结果如下:
1.缺血/再灌注抑制QKI-5表达
我们采用一个能够检测所有QKI亚型的抗体检测了乳鼠心肌细胞、H9C2细胞和成年大鼠心肌中的QKI表达。采用western blotting方法检测到了40 kDa和38 kDa两条带,按照分子量这两条带应该是QKI5和QKI6。另外采用间接免疫荧光方法检测了QKI的亚细胞定位,发现心肌细胞中QKI主要表达于细胞核,在胞浆中也可淡染。已有证据表明QKI5存在于细胞核,QKI6存在于胞浆和胞核,QKI7只存在于胞浆,故以上的数据提示心肌细胞中主要表达QKI5和QKI6。在NCM和H9C2细胞中,单纯培养基撤除可以抑制QKI5的表达,彻底缺血(培养基和氧供都撤除)进一步抑制QKI5表达,而缺血再灌注可以将QKI5的表达几乎清除。这些现象在免疫荧光实验中也得到了证实。与之不同的是,QKI6在这些处理中都没有发生明显改变。整体动物缺血/再灌注模型中,QKI5和QKI6都发生了表达下降,可能是由于心肌组织中细胞成分过多的原因。
2.沉默QKI表达增加H9C2细胞凋亡易感性
为了评价IR条件下QKI表达下降的意义,我们在H9C2细胞中采用RNA干涉沉默QKI表达。然后转染了阴性对照RNA(ncRNA)或者siRNA的细胞再用9 hr缺血加6小时再灌注处理。阴性对照未经任何处理,凋亡率为1.8±0.6;感染ncRNA细胞凋亡率为33.2±2.4 %,QKI干涉后细胞凋亡率为54.9±3.5 %,显著高于ncRNA组(P<0.01,图2.2.2)。
3.过表达QKI5或者QKI6抑制IR诱导的心肌细胞凋亡
乳鼠心肌细胞感染对照或者QKI5、6病毒,然后给与4 hr缺血加6 hr再灌注处理,采用Annexin V和PI双染经流式细胞仪检测凋亡率。4.9±1.6%的细胞凋亡;Ad-CMV组细胞凋亡率为34.0±2.3 %,Ad-QKI6组为14.7±2.2 %,另外,PARP实验还证实Ad-QKI5组凋亡显著低于Ad-EGFP组。
4.沉默QKI增加IR条件下FOXO1的表达
在不给予IR处理时,FOXO1表达量很低,沉默IR并没有影响FOXO1的表达,可见QKI的降低不是FOXO1升高的诱导因素。ncRNA组细胞中,IR可以使FOXO1表达显著升高;而在siRNA组细胞中,IR诱导的FOXO1增高更加显著。因此我们推测,IR过程中的其他因素诱导了FOXO1的表达,而QKI的降低导致了FOXO1表达的失控。为了证明QKI对FOXO具有负性调控作用,我们做了以下实验。
5.过表达QKI5或者QKI6下调FOXO1表达水平,并促其出核
在正常情况下,FOXO1表达水平很低,过表达QKI并没有影响其表达。IR诱导FOXO1大量表达,而过表达QKI5或者QKI6之后,将FOXO1的表达抑制到本底水平。间接免疫荧光实验也表明,QKI5或者QKI6过表达能够将细胞核中的FOXO1表达抑制到原始水平。胞浆中仍然能够观察到FOXO1表达,与未处理时水平相近。
总之,本研究首次提供证据证明QKI5和QKI6蛋白具有很强的抗缺血、再灌注所致心肌细胞凋亡的作用,这种作用可能通过抑制促凋亡转录因子FOXO1产生。另外,本研究还提示,在模拟缺血/再灌注时QKI5的降低导致了对促凋亡转录因子FOXO1的表达失去控制,最终(至少是部分)导致细胞凋亡的发生。
Acute cardiac infarction caused by complete coronary obstruction proposes one of the biggest threats on human life. To now, promptly and effectively restoring coronary blood flow is believed to be the most successful strategy to reduce the injury. However, such reperfusion could by itself induce cell death and approximately accumulate 50 % of the final infarct, which is termed myocardial reperfusion (IR) injury. This phenomenon may explain the high death rate and incidence of heart failure after acute myocardial infarction, despite optimal myocardial reperfusion was carried out in most cases. Actually, ischemia/ reperfusion damage in the myocardium is a messy affair with complex network of molecules and pathways involved in the competition between attack and self-protection as evidenced by a mass of papers. Hence, identification and characterization of the essential molecule contributing to IR injury or its counterpart would be critical to establish strategies maximizing the benefit of myocardial reperfusion both in basic research and under clinical settings. Here, we choose two molecule, scaffolding protein Homer 1 and RNA binding protein QKI, to explore their possible roles during myocardial ischemia/reperfusion.
I.Homer 1 protein Homer proteins are scaffolding proteins expressed in brain, heart and skeletal muscle, kidney, etc. All Homer isoforms bind to proteins containing a proline-rich motif (PPxxFr) through their N-terminal EVH1 domain, which is required for protein–protein interaction. All long Homer isoforms can form homo- or heteromultimers with themselves and other Homer family members, respectively, through the C-terminal CC domain containing leucine zipper motifs. Because of their distinct dimerization properties, long forms of Homer function both as scaffolds of multiprotein complexes and mediators of signaling pathways. Accumulated evidences in nervous system show that activation of ERK is dependent on Homer 1b/c and Homer 1a could interfer the ERK activation. In contrast, short forms of Homer, such as Homer1a, lack the dimerization domain and behave as dominant negatives. Homer isoforms, especially Homer 1, were proved to be abundantly expressed in myocardium, but there function is still not clear. In this study, we ask what the expression patterns of Homer 1 variants are during myocardial ischemia/reperfusion; what does such expression patterns mean to the ERK activation, cell death and the final cardiac function. The main results are as follows:
1. Ischemia/reperfusion induced Homer 1a accumulation in vitro and in vivo.
It has been reported that Homer 1a and 1b/c transcripts exist in heart, and proteins were detected with respective antibodies. We used commercial antibodies to determine Homer 1 expression in neonatal cardiomyocytes (NCM), H9C2 cells. Homer 1b/c and 1a could be both detected under normal culture conditions, but Homer 1a is only expressed with neural activity. We performed mimicked IR in NCM and H9C2 cells. Under culture medium deprivation, mimicked ischemia or ischemia/reperfusion (IR), Homer 1b/c expression remained unchanged, but Homer 1a could be up-regulated by these stimuli, among which IR induced the tiptop of Homer 1a expression.
2. Silencing Homer 1a elevated susceptibility to IR-induced apoptosis of H9C2 cells.
To evaluate what it means to the cardiomyocytes that Homer 1a accumulated under IR condition, we designed siRNAs to knockdown Homer 1a in H9C2 cells. Then the cells were subjected to 9-hour ischemia plus 6-hour reperfusion, apoptosis was determined by flow cytometry. Apoptosis rate was 43.6±3.4 % in ncRNA cells, 21.2±3.3 % in siRNA-a (P<0.05 vs ncRNA). These data indicated that Homer 1a up-regulation enhanced IR-induced apoptosis.
3. Over-expression of Homer 1a promoted IR-induced apoptosis in primary cardiomyocytes
To confirm whether Homer 1a expression promoted apoptosis, adeno- Homer 1a was constructed to investigate the direct effects of Homer 1a on IR- induced apoptosis in NCM. Apoptosis was determined by flowcytometry assay after the infected cells were subjected to IR stress. IR induced 52.8±2.3 % apoptosis in adeno-Homer 1a determined by flow cytometry, notably higher than that in cells with adeno-RFP 31.7±4.5 (%).
4. Silencing Homer 1a enhanced IR-induced ERK1/2 phosphorylation in H9C2 cells
ERK1/2 have been identified as kinases that could propose protection to heart, and constitutively activating MEK1/2 (ERK1/2 upstream kinases) could reduce IR-induced infarction. IR-induced ERK1/2 activation was determined after the genes were silenced. Four-hour ischemia plus 20-min reperfusion could substantially activate ERK1/2, which was exaggerated by knockdown of Homer 1a.
5. Over-expression of Homer 1a inhibited ERK1/2 activation during IR
To examine whether Homer 1a proposes an inhibition on ERK1/2 activation, phosphorylation of ERK1/2 in NCMs that had been over-expressed with Homer 1a by adenovirus was subjected to 4-hour ischemia and 20-min reperfusion. The IR treatment induced substantial increase of ERK1/2 phosphorylation in cells with control virus, while the induction was greatly suppressed in cells with adeno-Homer 1a.
The above observations provide the first evidence that expression of Homer 1a was induced by ischemia/reperfusion, which promoted ischemia/reperfusion in cardiac myocytes, possibly via the inhibition of anti-apoptotic ERK1/2 cascade.
II.RNA binding protein QKI
QKI belongs to signal transduction and activation of RNA (STAR) family protein. The major QKI isoforms include QKI-5, QKI-6 and QKI-7, which are derived from the qkI primary transcript via extensive alternative splicing of the C-terminal coding exons; each harbors a single hnRNP K-homology (KH) RNA-binding domain. QKI proteins are abundantly expressed in brain and heart; they take effects by regulating metabolism of messenger RNA.The role of QKI in nervous systerm has been better defined, which involves controling mRNA homeostasis during myelinogenesis, and the deficiency of QKI results in misregulation of its RNA targets, which in turn leads to hypomyelination. But the functions of QKI in myocardium are still obscure. Because QKI could work as RNA binding protein by recgnizing target RNA 3’UTR specificially, we analysized a mass of possible targets of QKI, among which was the proapototic FOXO transcriptional factors that have 3 conservative QKI binding sites in the 3’UTR of mRNA. Hence, we observed the roles of QKI in ischemia/ reperfusion-incuced apoptosis and possible mechanism involving pro-apoptotic transcriptional factor FOXO1. The main results are as follows:
1. Mimicked ischemia/reperfusion suppressed QKI-5 expression
In our study a pan-QKI monoclonal antibody was used to determine QKI expression in neonatal cardiomyocytes (NCM), H9C2 cells and adult myocardium, detecting two bands of 40-kDa and 38-kDa with western blotting. Then we investigated the subcellular localization of QKI protein with immunofluorescence, showing that QKI is dominantly present in nucleus and slightly stained in cytoplasm. Since QKI-5 is only present in nucleus, QKI-7 in cytoplasm, and QKI-6 in both, our results suggest the major QKI isoforms are QKI-5 and -6 in cardiomyocytes. Concomitantly, QKI expressions under mimicked ischemia/reperfusion were also investigated by western blotting and immunofluorescence. In NCM and H9C2 cells, ischemia was mimicked by replacement culture medium with Tyrode’s buffer and deprivation of oxygen, while reperfusion was simulated by restoration of culture medium and oxygen. Deprivation of culture medium alone could inhibit QKI-5 expression slightly, but have no effects on QKI-6 expression. Complete ischemia (medium and oxygen withdrawal) for 4 hours decreased QKI-5 expression sharply. Restoring nutrients and oxygen not only failed to resume QKI-5 expression, but further suppressed it nearly to zero. The phenomenon was also observed in immunofluorescence assay. In contrast, QKI-6 expression was not influenced by all these treatment. QKI-5 and QKI-6 expressions were all suppressed by ischemia/reperfusion, which might be attributed to the multiple cell constitution.
2. Silencing QKI expression elevated the sensitivity to mimicked ischemia/ reperfusion-induced apoptosis in H9C2 cells
To evaluate what it means to the cardiomyocytes that QKI expression declines under IR condition, we performed RNA interference to silence QKI expression in H9C2 cells. After subjected to 9-hour ischemia plus 6-hour reperfusion, cells with QKI siRNA present an apoptosis rate of 54.9±3.5 % as showed by flow cytometry, significantly higher than that in negative control cells 33.2±2.4%(P<0.01). These results suggest QKI presence is critical for defense of cardiomyocytes.
3. Over-expression of QKI-5 and QKI-6 inhibited mimicked ischemia/ reperfusion-induced apoptosis in primary cardiomyocytes
Adenoviruses expressing QKI-5 and -6 were constructed to investigate the direct effects of QKI on IR-induced apoptosis. NCMs were infected with the viruses, which harvested adequate infection efficiency and expression efficacy. Apoptosis was determined by flowcytometry and PARP assay after the infected cells were subjected to IR stress. IR induced an apoptosis rate of 14.7±2.2 % in the cells infected with adeno-QKI-6, notably lower than that in cells with adeno-CMV (34.0±2.3 %, P<0.01). PARP assay confirmed the results in flowcytometry. Besides, PARP assay showed adeno-QKI-5 could also inhibit IR-induced apoptosis as indicated by reduced PARP degradation.
4. Silencing QKI exaggerated the induction of FOXO1 expression by ischemia/ reperfusion
Although QKI knockdown increased apoptotic sensitivity of H9C2 cells, it is still unknown whether FOXO1 was involved in the increase of apoptotic sensitivity. We found silencing QKI did not affect FOXO1 expression under normal conditions. IR could dramatically elevate FOXO1 expression, while the FOXO1 elevation was more prominent in cells with QKI siRNA. Hence, it is reasonable to postulate that there exists a negative regulation of FOXO1 by QKI.
5. Over-expression of QKI-5 and -6 decreased FOXO1 expression level and promoted its exclusion from nucleus
To confirm the existence of QKI-mediated negative regulation on FOXO1 expression, we determined the effects of aden-QKI-5 and -6 on FOXO1 expression and subcellular localization. As mentioned above, FOXO1 was expressed at a relative low level under normal conditions, which was not influenced by over-expressing QKI-5 or -6. IR greatly raised expression of FOXO1 in cells infected with control virus; however, IR failed to induce FOXO1 up-regulation in cells infected with QKI-5 or -6. In addition, immunofluorescence was applied to determine the subcellular distribution of FOXO1. FOXO1 was slightly stained both in cytoplasm and nucleus without IR stress. While facing IR, the nucleus FOXO1 expression was greatly elevated in cells with adeno-CMV. In contrast, addition of adeno-QKI5 failed IR in the induction of FOXO1 expression in nucleus.
The above observations provide the first evidence that expression of QKI-5 and -6 proposed potent anti-apoptotic effects against ischemia/reperfusion in cardiac myocytes, possibly via the inhibition of pro-apoptotic transcriptional factor. Besides, the current study suggests the decrease of QKI-5 of cardiomyocytes during mimicked IR led to loss of control of pro-apoptotic factors and subsequent apoptosis at least partially.
研究一、Homer 1脚架蛋白Homer在脑、心脏和骨骼肌,肾脏等组织表达。已有研究主要集中于神经系统,所有Homer蛋白都通过其N -末端EVH1域与其他含有脯氨酸富集的基序(PPxxFr)的分子相互作用。所有长Homer亚型可以通过C -末端循环域含有亮氨酸拉链基序与同族或其他Homer家庭成员形成同源、异源二聚体。由于其独特的二聚性能,长型的Homer作为支架蛋白调节多种信号通路;与此相反,短型的Homer,如Homer1a,缺乏二聚体形成结构域,常抑制长型行使的功能。神经系统的研究表明,ERK的激活依赖于Homer 1b/c,而Homer1a可以干扰ERK激活。ERK在心肌中可以抑制心肌细胞凋亡,在再灌注过程中发挥一种心肌保护效应。本研究试图观察Homer 1各亚型在心肌缺血/再灌注中的表达模式,探明其对缺血再灌注所致心肌细胞凋亡的影响及其可能的机制。主要结果如下:
1.离体和在体实验都证明缺血/再灌注可以诱导Homer1a上调
我们采用商业抗体检测了新生大鼠心肌细胞、H9C2细胞中Homer 1各亚型的表达情况。神经系统中,Homer 1a只有在神经元活动或者受到刺激时才表达。而在我们的观察中,正常心肌细胞可以表达Homer 1a和1b/c,与神经系统略有不同。在培养的新生大鼠心肌细胞和胚心来源的成肌细胞H9C2细胞中采用培养基和氧气同时撤除的方式模拟细胞缺血,Homer 1a都被缺血/再灌注大量诱导表达。与Homer 1a不同,Homer 1b/c在以上处理中均保持稳定。
2.沉默Homer 1a增加H9C2的凋亡敏感性
为了评价Homer 1a在缺血/再灌注条件下上调的意义,我们设计了针对Homer 1a的siRNA以敲除Homer 1a的表达。敲除之后的细胞再给予9 hr缺血和6 hr再灌注处理,采用流式细胞仪检测细胞凋亡率。转染了阴性对照RNA的细胞凋亡率为43.6±3.4 %,而转染了siRNA的细胞凋亡率为21.2±3.3 %,显著低于ncRNA组(P<0.05)。这些数据提示IR时Homer 1a的上调促进了心肌细胞凋亡。
3.过表达Homer 1a增加乳鼠心肌细胞凋亡易感性
为了确认Homer 1a是否促进心肌细胞凋亡,我们构建了表达Homer 1a的腺病毒。采用对照病毒(Ad-RFP)或者Homer 1a病毒(Ad-H1a)感染心肌细胞,然后再给予4 hr缺血和6 hr再灌注处理,采用Annexin V单染,流式细胞仪检测凋亡率,未经处理的细胞由于对胰酶的敏感,也产生了3.8±0.8 %的凋亡;感染Ad-RFP的细胞受到上述I4R6处理之后凋亡率为31.7±4.5 %;而感染Ad-Homer的细胞经I4R6处理之后凋亡率为52.8±2.3 %,显著高于Ad-RFP组。
4.沉默Homer 1a增加H9C2细胞中IR诱导的ERK1/2激活
ERK1/2是一种具有心肌保护作用的激酶,过表达组成性活化的MEK1/2(ERK1/2上游激酶)可以缩小IR引起的心肌梗死范围。沉默Homer 1a之后,4 hr缺血加6小时再灌注都不能诱导Homer 1a增高。4 hr缺血和20 min再灌注之后,ERK1/2被激活,而在Homer 1a沉默的细胞中,ERK1/2激活的程度更加显著。
5.过表达Homer 1a抑制IR过程中ERK1/2的激活
为了检验Homer 1a对心肌中ERK1/2的激活是否有抑制作用,腺病毒感染乳鼠心肌细胞后给予4 hr缺血加20 min再灌注。我们发现,IR在对照病毒感染的细胞中诱导了ERK1/2的显著激活;而采用腺病毒感染过表达Homer 1a不仅可以抑制ERK1/2的本底磷酸化水平,还可以抑制IR诱导的ERK1/2激活。
总之,以上研究首次发现IR可以诱导Homer 1a表达上调,这种上调促进心肌细胞凋亡,可能的机制为Homer 1a的上调抑制了心肌保护激酶ERK1/2的充分激活。
研究二、QKI蛋白QKI属于信号转导和RNA激活(STAR)家族蛋白,包括QKI-5、-6、-7三种异构体,qkI的初始转录本C末端外显子选择性剪接后表达。每一种QKI异构体都含有KH RNA结合结构域。QKI蛋白在脑和心脏大量表达并通过调节mRNA代谢发挥作用。QKI在神经系统中的作用得到了广泛的研究,证明其可以通过调控参与髓鞘生成过程中相关mRNA的稳态而参与髓鞘生成,QKI缺乏可以导致髓鞘生成障碍。由于QKI蛋白通过特异结合其靶mRNA的3’UTR起作用,通过生物信息学分析,有很多mRNA可能是QKI蛋白的靶分子,其中促凋亡蛋白Foxo 1 mRNA的3’UTR有3个QKI保守结合位点。因此我们致力于研究QKI蛋白在心肌细胞凋亡中的作用,并试图探明其与FOXO1之间的调控关系。主要结果如下:
1.缺血/再灌注抑制QKI-5表达
我们采用一个能够检测所有QKI亚型的抗体检测了乳鼠心肌细胞、H9C2细胞和成年大鼠心肌中的QKI表达。采用western blotting方法检测到了40 kDa和38 kDa两条带,按照分子量这两条带应该是QKI5和QKI6。另外采用间接免疫荧光方法检测了QKI的亚细胞定位,发现心肌细胞中QKI主要表达于细胞核,在胞浆中也可淡染。已有证据表明QKI5存在于细胞核,QKI6存在于胞浆和胞核,QKI7只存在于胞浆,故以上的数据提示心肌细胞中主要表达QKI5和QKI6。在NCM和H9C2细胞中,单纯培养基撤除可以抑制QKI5的表达,彻底缺血(培养基和氧供都撤除)进一步抑制QKI5表达,而缺血再灌注可以将QKI5的表达几乎清除。这些现象在免疫荧光实验中也得到了证实。与之不同的是,QKI6在这些处理中都没有发生明显改变。整体动物缺血/再灌注模型中,QKI5和QKI6都发生了表达下降,可能是由于心肌组织中细胞成分过多的原因。
2.沉默QKI表达增加H9C2细胞凋亡易感性
为了评价IR条件下QKI表达下降的意义,我们在H9C2细胞中采用RNA干涉沉默QKI表达。然后转染了阴性对照RNA(ncRNA)或者siRNA的细胞再用9 hr缺血加6小时再灌注处理。阴性对照未经任何处理,凋亡率为1.8±0.6;感染ncRNA细胞凋亡率为33.2±2.4 %,QKI干涉后细胞凋亡率为54.9±3.5 %,显著高于ncRNA组(P<0.01,图2.2.2)。
3.过表达QKI5或者QKI6抑制IR诱导的心肌细胞凋亡
乳鼠心肌细胞感染对照或者QKI5、6病毒,然后给与4 hr缺血加6 hr再灌注处理,采用Annexin V和PI双染经流式细胞仪检测凋亡率。4.9±1.6%的细胞凋亡;Ad-CMV组细胞凋亡率为34.0±2.3 %,Ad-QKI6组为14.7±2.2 %,另外,PARP实验还证实Ad-QKI5组凋亡显著低于Ad-EGFP组。
4.沉默QKI增加IR条件下FOXO1的表达
在不给予IR处理时,FOXO1表达量很低,沉默IR并没有影响FOXO1的表达,可见QKI的降低不是FOXO1升高的诱导因素。ncRNA组细胞中,IR可以使FOXO1表达显著升高;而在siRNA组细胞中,IR诱导的FOXO1增高更加显著。因此我们推测,IR过程中的其他因素诱导了FOXO1的表达,而QKI的降低导致了FOXO1表达的失控。为了证明QKI对FOXO具有负性调控作用,我们做了以下实验。
5.过表达QKI5或者QKI6下调FOXO1表达水平,并促其出核
在正常情况下,FOXO1表达水平很低,过表达QKI并没有影响其表达。IR诱导FOXO1大量表达,而过表达QKI5或者QKI6之后,将FOXO1的表达抑制到本底水平。间接免疫荧光实验也表明,QKI5或者QKI6过表达能够将细胞核中的FOXO1表达抑制到原始水平。胞浆中仍然能够观察到FOXO1表达,与未处理时水平相近。
总之,本研究首次提供证据证明QKI5和QKI6蛋白具有很强的抗缺血、再灌注所致心肌细胞凋亡的作用,这种作用可能通过抑制促凋亡转录因子FOXO1产生。另外,本研究还提示,在模拟缺血/再灌注时QKI5的降低导致了对促凋亡转录因子FOXO1的表达失去控制,最终(至少是部分)导致细胞凋亡的发生。
Acute cardiac infarction caused by complete coronary obstruction proposes one of the biggest threats on human life. To now, promptly and effectively restoring coronary blood flow is believed to be the most successful strategy to reduce the injury. However, such reperfusion could by itself induce cell death and approximately accumulate 50 % of the final infarct, which is termed myocardial reperfusion (IR) injury. This phenomenon may explain the high death rate and incidence of heart failure after acute myocardial infarction, despite optimal myocardial reperfusion was carried out in most cases. Actually, ischemia/ reperfusion damage in the myocardium is a messy affair with complex network of molecules and pathways involved in the competition between attack and self-protection as evidenced by a mass of papers. Hence, identification and characterization of the essential molecule contributing to IR injury or its counterpart would be critical to establish strategies maximizing the benefit of myocardial reperfusion both in basic research and under clinical settings. Here, we choose two molecule, scaffolding protein Homer 1 and RNA binding protein QKI, to explore their possible roles during myocardial ischemia/reperfusion.
I.Homer 1 protein Homer proteins are scaffolding proteins expressed in brain, heart and skeletal muscle, kidney, etc. All Homer isoforms bind to proteins containing a proline-rich motif (PPxxFr) through their N-terminal EVH1 domain, which is required for protein–protein interaction. All long Homer isoforms can form homo- or heteromultimers with themselves and other Homer family members, respectively, through the C-terminal CC domain containing leucine zipper motifs. Because of their distinct dimerization properties, long forms of Homer function both as scaffolds of multiprotein complexes and mediators of signaling pathways. Accumulated evidences in nervous system show that activation of ERK is dependent on Homer 1b/c and Homer 1a could interfer the ERK activation. In contrast, short forms of Homer, such as Homer1a, lack the dimerization domain and behave as dominant negatives. Homer isoforms, especially Homer 1, were proved to be abundantly expressed in myocardium, but there function is still not clear. In this study, we ask what the expression patterns of Homer 1 variants are during myocardial ischemia/reperfusion; what does such expression patterns mean to the ERK activation, cell death and the final cardiac function. The main results are as follows:
1. Ischemia/reperfusion induced Homer 1a accumulation in vitro and in vivo.
It has been reported that Homer 1a and 1b/c transcripts exist in heart, and proteins were detected with respective antibodies. We used commercial antibodies to determine Homer 1 expression in neonatal cardiomyocytes (NCM), H9C2 cells. Homer 1b/c and 1a could be both detected under normal culture conditions, but Homer 1a is only expressed with neural activity. We performed mimicked IR in NCM and H9C2 cells. Under culture medium deprivation, mimicked ischemia or ischemia/reperfusion (IR), Homer 1b/c expression remained unchanged, but Homer 1a could be up-regulated by these stimuli, among which IR induced the tiptop of Homer 1a expression.
2. Silencing Homer 1a elevated susceptibility to IR-induced apoptosis of H9C2 cells.
To evaluate what it means to the cardiomyocytes that Homer 1a accumulated under IR condition, we designed siRNAs to knockdown Homer 1a in H9C2 cells. Then the cells were subjected to 9-hour ischemia plus 6-hour reperfusion, apoptosis was determined by flow cytometry. Apoptosis rate was 43.6±3.4 % in ncRNA cells, 21.2±3.3 % in siRNA-a (P<0.05 vs ncRNA). These data indicated that Homer 1a up-regulation enhanced IR-induced apoptosis.
3. Over-expression of Homer 1a promoted IR-induced apoptosis in primary cardiomyocytes
To confirm whether Homer 1a expression promoted apoptosis, adeno- Homer 1a was constructed to investigate the direct effects of Homer 1a on IR- induced apoptosis in NCM. Apoptosis was determined by flowcytometry assay after the infected cells were subjected to IR stress. IR induced 52.8±2.3 % apoptosis in adeno-Homer 1a determined by flow cytometry, notably higher than that in cells with adeno-RFP 31.7±4.5 (%).
4. Silencing Homer 1a enhanced IR-induced ERK1/2 phosphorylation in H9C2 cells
ERK1/2 have been identified as kinases that could propose protection to heart, and constitutively activating MEK1/2 (ERK1/2 upstream kinases) could reduce IR-induced infarction. IR-induced ERK1/2 activation was determined after the genes were silenced. Four-hour ischemia plus 20-min reperfusion could substantially activate ERK1/2, which was exaggerated by knockdown of Homer 1a.
5. Over-expression of Homer 1a inhibited ERK1/2 activation during IR
To examine whether Homer 1a proposes an inhibition on ERK1/2 activation, phosphorylation of ERK1/2 in NCMs that had been over-expressed with Homer 1a by adenovirus was subjected to 4-hour ischemia and 20-min reperfusion. The IR treatment induced substantial increase of ERK1/2 phosphorylation in cells with control virus, while the induction was greatly suppressed in cells with adeno-Homer 1a.
The above observations provide the first evidence that expression of Homer 1a was induced by ischemia/reperfusion, which promoted ischemia/reperfusion in cardiac myocytes, possibly via the inhibition of anti-apoptotic ERK1/2 cascade.
II.RNA binding protein QKI
QKI belongs to signal transduction and activation of RNA (STAR) family protein. The major QKI isoforms include QKI-5, QKI-6 and QKI-7, which are derived from the qkI primary transcript via extensive alternative splicing of the C-terminal coding exons; each harbors a single hnRNP K-homology (KH) RNA-binding domain. QKI proteins are abundantly expressed in brain and heart; they take effects by regulating metabolism of messenger RNA.The role of QKI in nervous systerm has been better defined, which involves controling mRNA homeostasis during myelinogenesis, and the deficiency of QKI results in misregulation of its RNA targets, which in turn leads to hypomyelination. But the functions of QKI in myocardium are still obscure. Because QKI could work as RNA binding protein by recgnizing target RNA 3’UTR specificially, we analysized a mass of possible targets of QKI, among which was the proapototic FOXO transcriptional factors that have 3 conservative QKI binding sites in the 3’UTR of mRNA. Hence, we observed the roles of QKI in ischemia/ reperfusion-incuced apoptosis and possible mechanism involving pro-apoptotic transcriptional factor FOXO1. The main results are as follows:
1. Mimicked ischemia/reperfusion suppressed QKI-5 expression
In our study a pan-QKI monoclonal antibody was used to determine QKI expression in neonatal cardiomyocytes (NCM), H9C2 cells and adult myocardium, detecting two bands of 40-kDa and 38-kDa with western blotting. Then we investigated the subcellular localization of QKI protein with immunofluorescence, showing that QKI is dominantly present in nucleus and slightly stained in cytoplasm. Since QKI-5 is only present in nucleus, QKI-7 in cytoplasm, and QKI-6 in both, our results suggest the major QKI isoforms are QKI-5 and -6 in cardiomyocytes. Concomitantly, QKI expressions under mimicked ischemia/reperfusion were also investigated by western blotting and immunofluorescence. In NCM and H9C2 cells, ischemia was mimicked by replacement culture medium with Tyrode’s buffer and deprivation of oxygen, while reperfusion was simulated by restoration of culture medium and oxygen. Deprivation of culture medium alone could inhibit QKI-5 expression slightly, but have no effects on QKI-6 expression. Complete ischemia (medium and oxygen withdrawal) for 4 hours decreased QKI-5 expression sharply. Restoring nutrients and oxygen not only failed to resume QKI-5 expression, but further suppressed it nearly to zero. The phenomenon was also observed in immunofluorescence assay. In contrast, QKI-6 expression was not influenced by all these treatment. QKI-5 and QKI-6 expressions were all suppressed by ischemia/reperfusion, which might be attributed to the multiple cell constitution.
2. Silencing QKI expression elevated the sensitivity to mimicked ischemia/ reperfusion-induced apoptosis in H9C2 cells
To evaluate what it means to the cardiomyocytes that QKI expression declines under IR condition, we performed RNA interference to silence QKI expression in H9C2 cells. After subjected to 9-hour ischemia plus 6-hour reperfusion, cells with QKI siRNA present an apoptosis rate of 54.9±3.5 % as showed by flow cytometry, significantly higher than that in negative control cells 33.2±2.4%(P<0.01). These results suggest QKI presence is critical for defense of cardiomyocytes.
3. Over-expression of QKI-5 and QKI-6 inhibited mimicked ischemia/ reperfusion-induced apoptosis in primary cardiomyocytes
Adenoviruses expressing QKI-5 and -6 were constructed to investigate the direct effects of QKI on IR-induced apoptosis. NCMs were infected with the viruses, which harvested adequate infection efficiency and expression efficacy. Apoptosis was determined by flowcytometry and PARP assay after the infected cells were subjected to IR stress. IR induced an apoptosis rate of 14.7±2.2 % in the cells infected with adeno-QKI-6, notably lower than that in cells with adeno-CMV (34.0±2.3 %, P<0.01). PARP assay confirmed the results in flowcytometry. Besides, PARP assay showed adeno-QKI-5 could also inhibit IR-induced apoptosis as indicated by reduced PARP degradation.
4. Silencing QKI exaggerated the induction of FOXO1 expression by ischemia/ reperfusion
Although QKI knockdown increased apoptotic sensitivity of H9C2 cells, it is still unknown whether FOXO1 was involved in the increase of apoptotic sensitivity. We found silencing QKI did not affect FOXO1 expression under normal conditions. IR could dramatically elevate FOXO1 expression, while the FOXO1 elevation was more prominent in cells with QKI siRNA. Hence, it is reasonable to postulate that there exists a negative regulation of FOXO1 by QKI.
5. Over-expression of QKI-5 and -6 decreased FOXO1 expression level and promoted its exclusion from nucleus
To confirm the existence of QKI-mediated negative regulation on FOXO1 expression, we determined the effects of aden-QKI-5 and -6 on FOXO1 expression and subcellular localization. As mentioned above, FOXO1 was expressed at a relative low level under normal conditions, which was not influenced by over-expressing QKI-5 or -6. IR greatly raised expression of FOXO1 in cells infected with control virus; however, IR failed to induce FOXO1 up-regulation in cells infected with QKI-5 or -6. In addition, immunofluorescence was applied to determine the subcellular distribution of FOXO1. FOXO1 was slightly stained both in cytoplasm and nucleus without IR stress. While facing IR, the nucleus FOXO1 expression was greatly elevated in cells with adeno-CMV. In contrast, addition of adeno-QKI5 failed IR in the induction of FOXO1 expression in nucleus.
The above observations provide the first evidence that expression of QKI-5 and -6 proposed potent anti-apoptotic effects against ischemia/reperfusion in cardiac myocytes, possibly via the inhibition of pro-apoptotic transcriptional factor. Besides, the current study suggests the decrease of QKI-5 of cardiomyocytes during mimicked IR led to loss of control of pro-apoptotic factors and subsequent apoptosis at least partially.
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