Wnt-5a/Frizzled-2途径对心肌缺血再灌注损伤后心肌Ca~(2+)变化的影响的研究
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摘要
研究背景：
心肌缺血再灌注损伤是指缺血期处于可逆损伤的心肌细胞经恢复血液供应后缺血心肌细胞的损伤反而加重,并转化为不可逆性。随着冠状动脉溶栓术、冠状动脉搭桥术等技术的推广应用,心肌缺血再灌注损伤成为阻碍缺血心肌从再灌注疗法中获得最佳疗效的主要难题。目前认为心肌缺血再灌注损伤发病机制主要有：能量代谢障碍、氧自由基产生、钙超载形成等,能量代谢是始动因素,氧自由基生成是造成缺血再灌注损伤的重要环节,钙超载是缺血再灌注的不可逆损伤的最后通路。
临床和实验研究已证实,在心肌缺血再灌注损伤后心肌细胞内存在钙超载,并且细胞内Ca2+超载在心肌缺血再灌注损伤发病机制中起主导作用,细胞内Ca2+平衡状态紊乱后引起Ca2+内流和Ca2+分隔机制的失调导致了缺血再灌注心肌细胞内Ca2+超载。钙离子作为第二信使,在细胞信息传递和损伤中起重要作用。静息状态下,细胞外游离Ca2+浓度为0.1-10mmol/L,而胞内仅为0.1μmol/L左右,主要分布在细胞核、线粒体、内质网/肌浆网和质膜上,当刺激使胞外即使少量的Ca2+进入胞内或钙库释放稍有增加时,均可导致胞浆内Ca2+浓度大幅度增加,继而发生一系列生理、生化反应,如细胞结构的损伤、凋亡、死亡和细胞的退行性改变等作用,因此调节细胞内钙离子的动态平衡对维持细胞正常的生理功能和信息传递十分重要。
据目前研究发现,缺血及再灌注时钙超载主要发生在再灌注期。钙超载可导致心室舒张不全,严重者可因心室充盈不足引起舒张性心力衰竭;钙超载能够引起酸中毒和促进心律失常的发生;再灌注时心肌出现的强烈收缩带、肌纤维断裂和坏死；钙超载可以激活蛋白酶和钙依赖性磷脂酶,破坏生物膜的结构完整性,并在膜磷脂分解过程中产生溶血磷脂进入线粒体抑制ATP的合成;加之大量Ca2+进入线粒体以磷酸钙的形式沉积于线粒体中,从而破坏了线粒体的氧化磷酸化功能。Sjaastad等研究,心肌梗死后存活心肌细胞更容易出现钙超载,因而更容易受缺血再灌注损伤。
已知细胞内钙是循环系统中重要的第二信使,调节不同的细胞功能,是维持细胞的正常状态的重要因素,是细胞之间及其与外界进行信息交流的重要介质。在正常状态下,构成线粒体膜电位的质子泵电子转运系统可将胞内多余钙离子泵出细胞外,以维持细胞内的正常钙储存水平。但在某些病理的情况下,比如心肌缺血再灌注损伤(MRI),细胞损伤后这种稳定状态就被打破,导致细胞内的钙离子积聚直至钙超载发生,最终导致细胞的死亡。
研究目的：
一些研究提出Wnt5a是通过激活Frizzled-2受体,导致Ca2+动员而发挥作用的。对斑马鱼和非洲蟾蜍的研究,为非经典Wnt/Ca2+途径提供了第一手的证据。在单个细胞斑马鱼胚胎中注射Wnt5a mRNA时发现胚层上钙变化频率增大两倍,这是第一次发现钙在Wnt信号途径中具有第二信使的作用。Frizzle-2是Wnt5a配体的细胞表面受体,在斑马鱼中异位表达大鼠Frizzled-2能使细胞内钙释放增加,研究提示Wnt5a经由Frizzled-2受体刺激钙离子释放。目前研究集中在阐述wnt和Frizzled在低等动物,比如非洲蟾蜍和斑马鱼细胞或组织中如何结合以及结合后下游信号途径的表达,但Wnt5a/Frizzled-2信号途径在哺乳动物心肌细胞中的是否存在,如果存在那么如何激活?激活后是否也可以引起细胞或组织中的Ca2+变化却知之甚少。
综合以上资料,钙离子超载是MRI的主要机制之一,Wnt5a/Frizzled-2信号途径又与细胞内的钙离子变化有关,那么两者之间有没有什么联系呢?Wnt5a/Frizzled-2信号途径在心肌缺血再灌注损伤这样特定病理环境中有没有被激活,在心肌损伤机制中又扮演着什么样的角色呢?
方法和结果：
本研究从以上几点假设为出发,经体外、体内以及细胞缺氧／复氧模拟体内缺血再灌注损伤内环境三部分研究,最终证明了Wnt5a/Frizzled-2信号途径在哺乳动物的心肌细胞中同样存在,且在心肌缺血再灌注损伤后能够激活并参与到了损伤后心肌细胞钙离子超载的过程中。
第一部分实验：我们首先培养了大鼠心肌H9c2细胞,而后使用QRT-PCR和Western-blot方法分别检测了正常大鼠H9c2细胞中Wnt5a和Frizzled-2的基因及蛋白质的表达,以及正常细胞中钙离子的含量,结果肯定了我们的假设和猜测,大鼠H9c2细胞中存在Frizzled-2和Wnt5a的表达,但是表达水平较低；接下来为了证明此途径在某种条件下被激活后,对细胞内的钙离子含量的影响,我们利用全基因合成的frizzled-2质粒,结合Lipo2000将其转染至正常的H9C2细胞内,发现转染后细胞中Wnt5a和Frizzled-2无论在基因还是蛋白水平上都发生了显著的增高,基因表达与未转染的正常细胞相比分别上调了12倍和10倍(P<0.001),蛋白质表达水平也分别增加了8倍和3倍(P<0.001),同时我们检测到了此途径激活特异性标志蛋白p-caMK Ⅱ的表达增加了4倍(P<0.001),为检测细胞内的钙离子浓度的变化情况,我们用共聚焦显微镜观察与fluo-3AM共孵育后H9c2细胞中Ca2+浓度,结果显示Ca2+随之也发生了显著的升高(P<0.001),结果具有统计学意义。这说明细胞内的钙离子水平的变化与此途径的激活有一定的相关性,但这条途径在某种条件下激活后导致了细胞内的钙离子水平增高,结论尚早。
针对这一问题,我们使用Stealth RNAi靶向沉默frizzled-2基因,抑制Frizzled-2蛋白质的表达,结果显示靶向抑制Frizzled-2表达后最终抑制Wnt5a/Frizzled-2信号途径的活性,降低了细胞内的钙离子水平。结果再次证实了我们的假设,与单纯质粒升高Frizzled-2的细胞相比,抑制Frizzled-2表达的细胞中Wnt5a与Frizzled-2基因及蛋白的表达均显著降低(P<0.001),与之呼应的是途径激活特异性标志蛋白p-caMK Ⅱ的表达减低1.5倍(P<0.001),同时细胞内的钙离子含量也显著的降低(P<0.001)。这说明,Wnt5a/Frizzled-2信号途径在某种条件下激活后,能够引起大鼠H9c2细胞中的钙离子含量增高。
第二部分实验：我们已知钙离子超载是缺血-再灌注损伤的重要机制之一。研究发现[33]在正常状态的成年心脏和血管中,Wnt信号表达水平非常低,但在压力导致的心脏重塑、血管损伤和心肌梗死后该途径激活。Blankesteijn[34]等人的研究发现在大鼠心肌梗死后的10天内均检测到。那么心肌缺血-再灌注会不会引起损伤心肌细胞中frizzled-2mRNA水平上调呢?缺血-再灌注会不会引起Wnt5a/Frizzled-2信号途径的激活呢?本部分的实验我们检测了大鼠心肌缺血-再灌注模型中信号途径的两个重要组分Frizzled-2和Wnt5a的变化情况。
研究采用QRT-PCR和Western-blot两种方法分别从基因和蛋白质水平检测Wnt5a和Frizzled-2在正常大鼠和缺血再灌注大鼠模型心肌组织中的表达情况。实验结果证实与体外实验相符,Wnt5a/Frizzled-2信号途径的两个重要成分Frizzled-2和Wnt5a同样存在于正常大鼠的心肌组织中。我们发现,损伤后Frizzled-2和Wnt5a的表达增高,基因水平分别上调了8倍和3倍(P<0.001,P<0.05),蛋白表达增高4倍和6倍(P<0.001)。通过对此途径激活的特异性标志蛋白p-caMKⅡ的表达检测发现,在心肌损伤后,损伤组织中p-caMKⅡ的表达量显著增高7倍(P<0.001),这说明Wnt5a/Frizzled-2信号途径可能在损伤后激活了,并且可能与损伤细胞中钙离子的增幅有关。
为了进一步证明Wnt5a/Frizzled-2信号途径的激活参与了心肌缺血再灌注损伤后心肌细胞的钙离子超载过程,我们选择使用沉默frizzled-2基因的方法来进行。由于利用基因敲除鼠来实验不仅费时费力,而且耗资巨大,所以我们针对frizzled-2设计了特异性的抑制剂Stealth RNAi,计划利用体内转染试剂将Stealth RNAi通过尾静脉或者靶向注射的方法沉默心肌组织中frizzled-2基因,观察Wnt5a和Frizzled-2的基因及蛋白在心肌组织损伤后组织中的表达情况。但是,这样安排需要面对两个问题：首先,这种合成的靶向抑制试剂非常的昂贵,而且剂量很少,加上本实验对动物的需求量大,如果使用尾静脉注射的方法将无法满足实验需求；其次,尾静脉注射的方法需要量虽大,但抑制剂对特定部位的抑制效率却不一定高,也就是说特异性不好；第三,如果选用靶向注射的方法,技术条件不仅不允许,而且容易对心脏造成一定的损害,会对本实验的研究目的造成影响。
第三部分实验：结合以上三点原因,我们设计使用体外细胞培养进行缺氧/复氧的方式来模拟缺血再灌注损伤后心肌细胞的伤后环境,进而证明心肌缺血再灌注后,Wnt5a/Frizzled-2信号途径的激活参与心肌细胞缺氧/复氧的钙离子超载。QRT-PCR检测结果显示,正常大鼠H9C2细胞缺氧/复氧后细胞中的frizzled-2和Wnt5a基因表达发生了显著的上调,分别上调了8倍和3倍(P<0.001,P<0.05),与正常细胞表达量相比增高显著；Western-blot检测结果显示,细胞经缺氧/复氧处理后,细胞中Wnt5a和Frizzled-2蛋白的表达较正常未处理细胞显著增高,分别增加了7倍和2倍(P<0.001)；激光共聚焦显微镜下检测结果发现,细胞经缺氧/复氧处理后,细胞中的钙离子荧光强度较正常细胞高,荧光值增大了2倍(P<0.001)。途径激活的特异性标志蛋白p-caMK Ⅱ随着Wnt5a和Frizzled-2的表达增高而增高显著(P<0.001)；这说明细胞经缺氧/复氧后,Wnt5a/Frizzled-2途径可能激活了,但与细胞中的钙离子含量增多之间有没有关联呢?
我们在对细胞转染Stealth RNAi预处理后,再进行缺氧/复氧后发现：Stealth RNAi对靶基因frizzled-2的抑制效率很高,与单纯缺氧/复氧细胞相比,当frizzled-2基因被抑制后,Wnt5a的特异性受体Frizzled-2的表达也被抑制,Wnt5a的基因和蛋白水平也随之下降显著(P<0.001),致使Wnt5a/Frizzled-2信号途径无法在细胞缺氧/复氧处理后激活,令人欣喜的是,在抑制细胞中Frizzled-2表达的后,缺氧/复氧后的细胞中的钙离子含量发生了显著的下降,激光共聚焦显微镜检测发现细胞中钙离子的荧光强度较单纯缺氧／复氧组细胞降低显著(P<0.001), p-caMK Ⅱ也降低了3倍(P<0.001)。
结论和意义：
从本研究结果我们可以得出以下结论：
(1) Wnt5a/Frizzled-2信号途径首先存在于哺乳动物的心肌细胞中；
(2)大鼠心肌缺血-再灌注损伤后心肌细胞中Frizzled-2和Wnt5a表达增加；
(3)体外模拟心肌缺血再灌注损伤能够激活H9c2大鼠心肌细胞中Wnt5a/Frizzled-2信号途径,后者对损伤后心肌细胞钙离子超载起到一定的作用。换句话说,Wnt5a/Frizzled-2信号途径在心肌缺血再灌注损伤后激活是心肌损伤后心肌细胞钙超载的可能机制之一。
(4)本研究验证了Stealth RNAi转染靶向沉默目的基因的可靠性,并且具有较高的抑制效率；
通过对Wnt5a/Frizzled-2信号途径在心肌缺血再灌注损伤过程中的作用研究,将有助于对损伤机制的理解,为研究、治疗提供新的可能的靶点。
Myocardial ischemia-reperfusion injury is reversible ischemic injury of myocardial cells transformed into irreversible when restoration of blood supply to myocardial cells. Now myocardial reperfusion injury become an obstacle for coronary thrombolysis and coronary artery bypass surgery application. As we know, the main mechenism of myocardial ischemia reperfusion injury are energy metabolism, free oxygen radicals and calcium overload formation. Energy metabolism is the initial factor, free oxygen radicals is an important part of ischemia-reperfusion injury, and calcium overload is a irreversible pathway in the final injury of ischemia-reperfusion.
Clinical and experimental studies have demonstrated that, calcium overload involved in myocardial ischemia-reperfusion injury, and plays a leading role in the pathogenesis of myocardial ischemia reperfusion injury. Calcium overload is induced by intracellular Ca2+equilibrium disorder. As a second messenger, calcium plays important roles in cell communication and injury. In resting state, the extracellular concentration of free Ca2+is0.1-10mmol/L, while the intracellular concentration is only0.1μmol/L, mainly distribute in the nucleus, mitochondria, endoplasmic reticulum/sarcoplasmic reticulum and the plasma membrane. When small amounts of extracellular Ca2+into the intracellular or calcium store release slightly increased, cytoplasmic Ca2+may significant increase, leading a series of physiological and biochemical reactions, such as cell structural damage, apoptosis, death and cell degeneration, etc., So maintaining intracellular calcium homeostasis is very important.
According to the present study that calcium overload occurs mainly in the reperfusion period. Calcium overload can lead to ventricular diastolic dysfunction, even caused diastolic heart failure; calcium overload can cause acidosis and arrhythmia; Strong contraction band, muscle fiber rupture and necrosis appear during reperfusion; The protease and calcium-dependent phospholipase can be activated by calcium overload and undermine the structural integrity of the biofilm, and produce lysophosphatidic in the membrane phospholipid decomposition process into the mitochondria to inhibite ATP synthesis; In addition, a large number of Ca2+in the form of calcium phosphate deposits in the mitochondria, damage the mitochondrial oxidative phosphorylation. Sjaastad's studies showed that survival cells after myocardial infarction more prone to take place calcium overload, and therefore more vulnerable to sufferischemia-reperfusion injury.
Intracellular calcium is an important second messenger in circulatory system, can regulate diverse cellular functions and maintain normal cells state, it is also a media for information exchange between cells and with the outside world. In the normal state, redundancy calcium can be pumped out by mitochondrial membrane potential proton pump electronic intracellular transport system to maintain normal intracellular calcium storage levels. However, in certain pathological cases, such as myocardial ischemia-reperfusion injury (MRI), stable state was broken up, resulting in the accumulation of intracellular calcium up to calcium overload, eventually leading to cell death.
Objective
Based on the above data, calcium overload is one of the main mechanism of MRI, and Wnt5a/Frizzled-2signaling pathway is related with intracellular calcium, then is there any connection between them? Whether Wnt5a/Frizzled-2signaling pathways activted after myocardial ischemia reperfusion injury, and involved in this Pathological process?
Methods and Results
According to our hypothesis, in vitro and in vivo models were used to vertify Wnt5a/Frizzled-2signaling patyway existed in myocardial cells and involved in the mechanism of myocardial ischemia reperfusion injury.
Part one:To determine whether Frizzled-2and Wnt5a were expressed in H9c2cells, we examined their expressions in non-transfected (normal) H9c2cells by Q-PCR and Western blot. We found that Frizzled-2and Wnt5a were weakly expressed through both Q-PCR and Western blot detection. Next, to examine the roles of Frizzled-2in Wnt5a signaling and calcium inflow, we transfected rat H9c2cells with synthesized frizzled-2plasmid and then analyzed the expression of Frizzle-2and Wnt5a collected from Q-PCR and Western blot. When the cells were transfected, we observed that gene expressions of both frizzled-2and Wnt5a were significantly increased approximately12-fold and10-fold, respectively, in comparison to that in non-transfected cells (P<0.001). Moreover, protein expressions of both Frizzled-2and Wnt5a were significantly increased approximately8-and3-fold, respectively, when compared with that in non-transfected cells (P<0.001). In addition, it is known that phosphorylation of CaMK II indicates the action of the frizzled-2mediate Wnt5a signaling pathway [35], and we found that p-CaMK Ⅱ was also significantly increased4-fold after the transfection (P<0.001;), compared to non-transfected cells.To identify whether activation of the Wnt5a/Frizzled-2pathway regulates Ca2+ release from intracellular stores, we examined whether changes in intracellular Ca2+inflow was affected by frizzled-2-transfection, indicated by the intensity of fluorescence of fluo-3/AM under confocal microscope after transfection. We found that the intensity was significantly increased2.5-fold after the transfection (P<0.001) when compared to the normal cells. This indicated that intracellular Ca2+inflow was increased in frizzled-2-transfected H9c2cells.
As described above, we showed that the expression of Wnt5a was closely associated with the expression of the Frizzled-2receptor, and that cells transfected with frizzled-2plasmid dramatically increased intracellular Ca2+inflow. Nevertheless, we could not draw a conclusion that Frizzled-2transduces binding of Wnt5a and leads to the intracellular Ca2+release. To resolve this problem, a special inhibitor called stealth RNAi was used to block the expression of the frizzled-2gene. Compared to the treatment without stealth RNAi, we found that the treatment with stealth RNAi significantly inhibited both gene and protein expression of Frizzled-2in transfected H9c2cells nearly to the control level, as demonstrated by Q-PCR and Western blot (P<0.001). Moreover, the expressions of the Wnt5a gene and protein were also markedly downregulated in transfected H9c2cells following the suppression of the frizzled-2gene by the treatment of stealth RNAi (P<0.001), compared to that without this treatment. To examine whether the expression of p-CaMKⅡ was blocked by the inhibition of frizzled-2signaling, we detected its expression by Western blot after application of stealth RNAi. We found that the treatment of stealth RNAi significantly inhibited the expression of p-caMK Ⅱ by1.5-fold in frizzled-2transfected H9c2cells, indicating that the activation of the Wnt5a/Frizzled-2pathway was suppressed by blocking frizzled-2.
To examine whether intracellular Ca2+inflow was affected by blocking frizzled-2signaling, we measured the intensity of fluorescence of fluo-3/AM under confocal microscope after application of stealth RNAi. We found that stealth RNAi dramatically decreased the intensity of fluorescence in frizzled-2transfected H9c2cells, compared to that without the treatment. Taken together, we can draw a conclusion that the Wnt5a/Frizzled-2pathway exists in rat H9c2cells and activates Ca2+release from intracellular stores.
Part two:Ca2+overload is known to be an important mechanism after I/R. In the adult heart and blood vessels, Wnt signaling activity is quite low under normal conditions. However, this pathway is reactivated during the pathological cardiac remodeling induced by pressure overload, in injured arteries and after myocardial infarction. In an initial study by Blankesteijn, et al, frizzled-2mRNA levels were found to be gradually upregulated in the first10days after myocardial infarction in the rat. Therefore, we used a rat I/R model to test whether the Wnt5a/Frizzled-2signaling pathway relative elements upregulate in injured cadiocyte. Firstly, we detected the expression of Frizzled-2and Wnt5a in normal rat H9c2cells on both the gene and protein level. We found that gene and protein expressions of both Frizzled-2and Wnt5a were expressed in normal cadiocyte. However, after I/R, gene expressions of frizzled-2and Wnt5a were significantly elevated approximately8-and3-fold, respectively, in the cadiocyte, when compared with that in normal H9c2cells (P<0.001and P<0.05, respectively). Protein expressions of Frizzled-2and Wnt5a were also elevated by4-and6-fold in cadiocyte, respectively, when compared to that in normal cadiocyte (P<0.001).While p-CaMKII was expressed at a very low level in normal cadiocyte, however, its expression was markedly increased approximately7-fold in the cadiocyte after I/R (P<0.001).
Because the mechanism of intracellular Ca2+accumulation after I/R are complicate, and hard to examine whether Wnt5a/Frizzled-2pathway contributes to the Ca2+accumulation after in vivo. So we choose to verify our hypothsis in pure populations of these cells.
Part three:we used H/R to mimic I/R in vivo [35,36]. Following H/R, We analyzed the expression of Frizzle-2and Wnt5a collected from Q-PCR and Western blot, observed that gene expressions of both frizzled-2and Wnt5a were significantly increased approximately8-fold and3-fold, respectively, in comparison to that normal cells (P<0.001). Moreover, protein expressions of both Frizzled-2and Wnt5a were significantly increased approximately7-and2-fold, respectively, when compared with that in normal cells (P<0.001).We found that the Ca2+intensity was significantly increased2-fold after H/R (P<0.001) when compared to nomal cells. This indicated that intracellular Ca2+inflow was increased in H/R treatment H9c2cells.In addition, we found that p-CaMK II was also significantly increased4-fold after the H/R (P<0.001), compared to non-transfected cells.
To determine the interaction between H/R-induced activation of Wnt5a/Frizzled-2signaling and accumulation of intracellular Ca2+, we suppressed the expression of the frizzled-2receptor gene by transfect with stealth RNAi before H/R, and detected the effects of stealth RNAi on gene and protein expression of Frizzled-2and Wnt5a after H/R. we observed a significant inhibitory effect on gene expression of frizzled-2in the transfected H9c2cells, compared with that in cells without transfect with stealth RNAi, as demonstrated by the fact that gene expression of frizzled-2was inhibited nearly6-fold. Surprisingly, the expression of the Wnt5a gene was also downregulated nearly3-fold (P<0.001). In addition, protein expressions of Frizzled-2and Wnt5a in transfected cells was significant decreased when compared to cells without transfection (P<0.001). Encouraging, the intensity of Ca2+fluorescence was significantly decreased in transfected cells after the H/R, when compared to those without transfection(P<0.001).Moreover, p-CaMKII in transfected cells was also inhibited by nearly3-fold, compare to only H/R treatment cells (P<0.001).
Conclusion and Significance
According to the results from this study we can draw the following conclusions:
(1) Wnt5a/Frizzled-2signaling pathways exist in mammals myocardial cells;
(2) Expression of Frizzled-2and Wnt5a were upregulated after myocardial ischemia reperfusion injury.
(3) We used hypoxia/reoxygenation to mimic myocardial ischemia-reperfusion injury in H9c2cells can activate Wnt5a/Frizzled-2signaling pathway. And found out that this pathway contribute to calcium overload after hypoxia/reoxygenation. In other words, Wnt5a/Frizzled-2signaling pathway was activated after hypoxia/reoxygenation, may may be one of the mechanisms of calcium overload after myocardial ischemia-reperfusion injury.
(3) This study tested the reliability of Stealth RNAi gene silencing, it has high inhibition efficiency;
By research on Wnt5a/Frizzled-2signaling pathways in myocardial ischemia reperfusion injury, may contribute to understanding the futher mechanism of reperfusion injury, will provide a new potential therapeutic target for treatment and research.
心肌缺血再灌注损伤是指缺血期处于可逆损伤的心肌细胞经恢复血液供应后缺血心肌细胞的损伤反而加重,并转化为不可逆性。随着冠状动脉溶栓术、冠状动脉搭桥术等技术的推广应用,心肌缺血再灌注损伤成为阻碍缺血心肌从再灌注疗法中获得最佳疗效的主要难题。目前认为心肌缺血再灌注损伤发病机制主要有：能量代谢障碍、氧自由基产生、钙超载形成等,能量代谢是始动因素,氧自由基生成是造成缺血再灌注损伤的重要环节,钙超载是缺血再灌注的不可逆损伤的最后通路。
临床和实验研究已证实,在心肌缺血再灌注损伤后心肌细胞内存在钙超载,并且细胞内Ca2+超载在心肌缺血再灌注损伤发病机制中起主导作用,细胞内Ca2+平衡状态紊乱后引起Ca2+内流和Ca2+分隔机制的失调导致了缺血再灌注心肌细胞内Ca2+超载。钙离子作为第二信使,在细胞信息传递和损伤中起重要作用。静息状态下,细胞外游离Ca2+浓度为0.1-10mmol/L,而胞内仅为0.1μmol/L左右,主要分布在细胞核、线粒体、内质网/肌浆网和质膜上,当刺激使胞外即使少量的Ca2+进入胞内或钙库释放稍有增加时,均可导致胞浆内Ca2+浓度大幅度增加,继而发生一系列生理、生化反应,如细胞结构的损伤、凋亡、死亡和细胞的退行性改变等作用,因此调节细胞内钙离子的动态平衡对维持细胞正常的生理功能和信息传递十分重要。
据目前研究发现,缺血及再灌注时钙超载主要发生在再灌注期。钙超载可导致心室舒张不全,严重者可因心室充盈不足引起舒张性心力衰竭;钙超载能够引起酸中毒和促进心律失常的发生;再灌注时心肌出现的强烈收缩带、肌纤维断裂和坏死；钙超载可以激活蛋白酶和钙依赖性磷脂酶,破坏生物膜的结构完整性,并在膜磷脂分解过程中产生溶血磷脂进入线粒体抑制ATP的合成;加之大量Ca2+进入线粒体以磷酸钙的形式沉积于线粒体中,从而破坏了线粒体的氧化磷酸化功能。Sjaastad等研究,心肌梗死后存活心肌细胞更容易出现钙超载,因而更容易受缺血再灌注损伤。
已知细胞内钙是循环系统中重要的第二信使,调节不同的细胞功能,是维持细胞的正常状态的重要因素,是细胞之间及其与外界进行信息交流的重要介质。在正常状态下,构成线粒体膜电位的质子泵电子转运系统可将胞内多余钙离子泵出细胞外,以维持细胞内的正常钙储存水平。但在某些病理的情况下,比如心肌缺血再灌注损伤(MRI),细胞损伤后这种稳定状态就被打破,导致细胞内的钙离子积聚直至钙超载发生,最终导致细胞的死亡。
研究目的：
一些研究提出Wnt5a是通过激活Frizzled-2受体,导致Ca2+动员而发挥作用的。对斑马鱼和非洲蟾蜍的研究,为非经典Wnt/Ca2+途径提供了第一手的证据。在单个细胞斑马鱼胚胎中注射Wnt5a mRNA时发现胚层上钙变化频率增大两倍,这是第一次发现钙在Wnt信号途径中具有第二信使的作用。Frizzle-2是Wnt5a配体的细胞表面受体,在斑马鱼中异位表达大鼠Frizzled-2能使细胞内钙释放增加,研究提示Wnt5a经由Frizzled-2受体刺激钙离子释放。目前研究集中在阐述wnt和Frizzled在低等动物,比如非洲蟾蜍和斑马鱼细胞或组织中如何结合以及结合后下游信号途径的表达,但Wnt5a/Frizzled-2信号途径在哺乳动物心肌细胞中的是否存在,如果存在那么如何激活?激活后是否也可以引起细胞或组织中的Ca2+变化却知之甚少。
综合以上资料,钙离子超载是MRI的主要机制之一,Wnt5a/Frizzled-2信号途径又与细胞内的钙离子变化有关,那么两者之间有没有什么联系呢?Wnt5a/Frizzled-2信号途径在心肌缺血再灌注损伤这样特定病理环境中有没有被激活,在心肌损伤机制中又扮演着什么样的角色呢?
方法和结果：
本研究从以上几点假设为出发,经体外、体内以及细胞缺氧／复氧模拟体内缺血再灌注损伤内环境三部分研究,最终证明了Wnt5a/Frizzled-2信号途径在哺乳动物的心肌细胞中同样存在,且在心肌缺血再灌注损伤后能够激活并参与到了损伤后心肌细胞钙离子超载的过程中。
第一部分实验：我们首先培养了大鼠心肌H9c2细胞,而后使用QRT-PCR和Western-blot方法分别检测了正常大鼠H9c2细胞中Wnt5a和Frizzled-2的基因及蛋白质的表达,以及正常细胞中钙离子的含量,结果肯定了我们的假设和猜测,大鼠H9c2细胞中存在Frizzled-2和Wnt5a的表达,但是表达水平较低；接下来为了证明此途径在某种条件下被激活后,对细胞内的钙离子含量的影响,我们利用全基因合成的frizzled-2质粒,结合Lipo2000将其转染至正常的H9C2细胞内,发现转染后细胞中Wnt5a和Frizzled-2无论在基因还是蛋白水平上都发生了显著的增高,基因表达与未转染的正常细胞相比分别上调了12倍和10倍(P<0.001),蛋白质表达水平也分别增加了8倍和3倍(P<0.001),同时我们检测到了此途径激活特异性标志蛋白p-caMK Ⅱ的表达增加了4倍(P<0.001),为检测细胞内的钙离子浓度的变化情况,我们用共聚焦显微镜观察与fluo-3AM共孵育后H9c2细胞中Ca2+浓度,结果显示Ca2+随之也发生了显著的升高(P<0.001),结果具有统计学意义。这说明细胞内的钙离子水平的变化与此途径的激活有一定的相关性,但这条途径在某种条件下激活后导致了细胞内的钙离子水平增高,结论尚早。
针对这一问题,我们使用Stealth RNAi靶向沉默frizzled-2基因,抑制Frizzled-2蛋白质的表达,结果显示靶向抑制Frizzled-2表达后最终抑制Wnt5a/Frizzled-2信号途径的活性,降低了细胞内的钙离子水平。结果再次证实了我们的假设,与单纯质粒升高Frizzled-2的细胞相比,抑制Frizzled-2表达的细胞中Wnt5a与Frizzled-2基因及蛋白的表达均显著降低(P<0.001),与之呼应的是途径激活特异性标志蛋白p-caMK Ⅱ的表达减低1.5倍(P<0.001),同时细胞内的钙离子含量也显著的降低(P<0.001)。这说明,Wnt5a/Frizzled-2信号途径在某种条件下激活后,能够引起大鼠H9c2细胞中的钙离子含量增高。
第二部分实验：我们已知钙离子超载是缺血-再灌注损伤的重要机制之一。研究发现[33]在正常状态的成年心脏和血管中,Wnt信号表达水平非常低,但在压力导致的心脏重塑、血管损伤和心肌梗死后该途径激活。Blankesteijn[34]等人的研究发现在大鼠心肌梗死后的10天内均检测到。那么心肌缺血-再灌注会不会引起损伤心肌细胞中frizzled-2mRNA水平上调呢?缺血-再灌注会不会引起Wnt5a/Frizzled-2信号途径的激活呢?本部分的实验我们检测了大鼠心肌缺血-再灌注模型中信号途径的两个重要组分Frizzled-2和Wnt5a的变化情况。
研究采用QRT-PCR和Western-blot两种方法分别从基因和蛋白质水平检测Wnt5a和Frizzled-2在正常大鼠和缺血再灌注大鼠模型心肌组织中的表达情况。实验结果证实与体外实验相符,Wnt5a/Frizzled-2信号途径的两个重要成分Frizzled-2和Wnt5a同样存在于正常大鼠的心肌组织中。我们发现,损伤后Frizzled-2和Wnt5a的表达增高,基因水平分别上调了8倍和3倍(P<0.001,P<0.05),蛋白表达增高4倍和6倍(P<0.001)。通过对此途径激活的特异性标志蛋白p-caMKⅡ的表达检测发现,在心肌损伤后,损伤组织中p-caMKⅡ的表达量显著增高7倍(P<0.001),这说明Wnt5a/Frizzled-2信号途径可能在损伤后激活了,并且可能与损伤细胞中钙离子的增幅有关。
为了进一步证明Wnt5a/Frizzled-2信号途径的激活参与了心肌缺血再灌注损伤后心肌细胞的钙离子超载过程,我们选择使用沉默frizzled-2基因的方法来进行。由于利用基因敲除鼠来实验不仅费时费力,而且耗资巨大,所以我们针对frizzled-2设计了特异性的抑制剂Stealth RNAi,计划利用体内转染试剂将Stealth RNAi通过尾静脉或者靶向注射的方法沉默心肌组织中frizzled-2基因,观察Wnt5a和Frizzled-2的基因及蛋白在心肌组织损伤后组织中的表达情况。但是,这样安排需要面对两个问题：首先,这种合成的靶向抑制试剂非常的昂贵,而且剂量很少,加上本实验对动物的需求量大,如果使用尾静脉注射的方法将无法满足实验需求；其次,尾静脉注射的方法需要量虽大,但抑制剂对特定部位的抑制效率却不一定高,也就是说特异性不好；第三,如果选用靶向注射的方法,技术条件不仅不允许,而且容易对心脏造成一定的损害,会对本实验的研究目的造成影响。
第三部分实验：结合以上三点原因,我们设计使用体外细胞培养进行缺氧/复氧的方式来模拟缺血再灌注损伤后心肌细胞的伤后环境,进而证明心肌缺血再灌注后,Wnt5a/Frizzled-2信号途径的激活参与心肌细胞缺氧/复氧的钙离子超载。QRT-PCR检测结果显示,正常大鼠H9C2细胞缺氧/复氧后细胞中的frizzled-2和Wnt5a基因表达发生了显著的上调,分别上调了8倍和3倍(P<0.001,P<0.05),与正常细胞表达量相比增高显著；Western-blot检测结果显示,细胞经缺氧/复氧处理后,细胞中Wnt5a和Frizzled-2蛋白的表达较正常未处理细胞显著增高,分别增加了7倍和2倍(P<0.001)；激光共聚焦显微镜下检测结果发现,细胞经缺氧/复氧处理后,细胞中的钙离子荧光强度较正常细胞高,荧光值增大了2倍(P<0.001)。途径激活的特异性标志蛋白p-caMK Ⅱ随着Wnt5a和Frizzled-2的表达增高而增高显著(P<0.001)；这说明细胞经缺氧/复氧后,Wnt5a/Frizzled-2途径可能激活了,但与细胞中的钙离子含量增多之间有没有关联呢?
我们在对细胞转染Stealth RNAi预处理后,再进行缺氧/复氧后发现：Stealth RNAi对靶基因frizzled-2的抑制效率很高,与单纯缺氧/复氧细胞相比,当frizzled-2基因被抑制后,Wnt5a的特异性受体Frizzled-2的表达也被抑制,Wnt5a的基因和蛋白水平也随之下降显著(P<0.001),致使Wnt5a/Frizzled-2信号途径无法在细胞缺氧/复氧处理后激活,令人欣喜的是,在抑制细胞中Frizzled-2表达的后,缺氧/复氧后的细胞中的钙离子含量发生了显著的下降,激光共聚焦显微镜检测发现细胞中钙离子的荧光强度较单纯缺氧／复氧组细胞降低显著(P<0.001), p-caMK Ⅱ也降低了3倍(P<0.001)。
结论和意义：
从本研究结果我们可以得出以下结论：
(1) Wnt5a/Frizzled-2信号途径首先存在于哺乳动物的心肌细胞中；
(2)大鼠心肌缺血-再灌注损伤后心肌细胞中Frizzled-2和Wnt5a表达增加；
(3)体外模拟心肌缺血再灌注损伤能够激活H9c2大鼠心肌细胞中Wnt5a/Frizzled-2信号途径,后者对损伤后心肌细胞钙离子超载起到一定的作用。换句话说,Wnt5a/Frizzled-2信号途径在心肌缺血再灌注损伤后激活是心肌损伤后心肌细胞钙超载的可能机制之一。
(4)本研究验证了Stealth RNAi转染靶向沉默目的基因的可靠性,并且具有较高的抑制效率；
通过对Wnt5a/Frizzled-2信号途径在心肌缺血再灌注损伤过程中的作用研究,将有助于对损伤机制的理解,为研究、治疗提供新的可能的靶点。
Myocardial ischemia-reperfusion injury is reversible ischemic injury of myocardial cells transformed into irreversible when restoration of blood supply to myocardial cells. Now myocardial reperfusion injury become an obstacle for coronary thrombolysis and coronary artery bypass surgery application. As we know, the main mechenism of myocardial ischemia reperfusion injury are energy metabolism, free oxygen radicals and calcium overload formation. Energy metabolism is the initial factor, free oxygen radicals is an important part of ischemia-reperfusion injury, and calcium overload is a irreversible pathway in the final injury of ischemia-reperfusion.
Clinical and experimental studies have demonstrated that, calcium overload involved in myocardial ischemia-reperfusion injury, and plays a leading role in the pathogenesis of myocardial ischemia reperfusion injury. Calcium overload is induced by intracellular Ca2+equilibrium disorder. As a second messenger, calcium plays important roles in cell communication and injury. In resting state, the extracellular concentration of free Ca2+is0.1-10mmol/L, while the intracellular concentration is only0.1μmol/L, mainly distribute in the nucleus, mitochondria, endoplasmic reticulum/sarcoplasmic reticulum and the plasma membrane. When small amounts of extracellular Ca2+into the intracellular or calcium store release slightly increased, cytoplasmic Ca2+may significant increase, leading a series of physiological and biochemical reactions, such as cell structural damage, apoptosis, death and cell degeneration, etc., So maintaining intracellular calcium homeostasis is very important.
According to the present study that calcium overload occurs mainly in the reperfusion period. Calcium overload can lead to ventricular diastolic dysfunction, even caused diastolic heart failure; calcium overload can cause acidosis and arrhythmia; Strong contraction band, muscle fiber rupture and necrosis appear during reperfusion; The protease and calcium-dependent phospholipase can be activated by calcium overload and undermine the structural integrity of the biofilm, and produce lysophosphatidic in the membrane phospholipid decomposition process into the mitochondria to inhibite ATP synthesis; In addition, a large number of Ca2+in the form of calcium phosphate deposits in the mitochondria, damage the mitochondrial oxidative phosphorylation. Sjaastad's studies showed that survival cells after myocardial infarction more prone to take place calcium overload, and therefore more vulnerable to sufferischemia-reperfusion injury.
Intracellular calcium is an important second messenger in circulatory system, can regulate diverse cellular functions and maintain normal cells state, it is also a media for information exchange between cells and with the outside world. In the normal state, redundancy calcium can be pumped out by mitochondrial membrane potential proton pump electronic intracellular transport system to maintain normal intracellular calcium storage levels. However, in certain pathological cases, such as myocardial ischemia-reperfusion injury (MRI), stable state was broken up, resulting in the accumulation of intracellular calcium up to calcium overload, eventually leading to cell death.
Objective
Based on the above data, calcium overload is one of the main mechanism of MRI, and Wnt5a/Frizzled-2signaling pathway is related with intracellular calcium, then is there any connection between them? Whether Wnt5a/Frizzled-2signaling pathways activted after myocardial ischemia reperfusion injury, and involved in this Pathological process?
Methods and Results
According to our hypothesis, in vitro and in vivo models were used to vertify Wnt5a/Frizzled-2signaling patyway existed in myocardial cells and involved in the mechanism of myocardial ischemia reperfusion injury.
Part one:To determine whether Frizzled-2and Wnt5a were expressed in H9c2cells, we examined their expressions in non-transfected (normal) H9c2cells by Q-PCR and Western blot. We found that Frizzled-2and Wnt5a were weakly expressed through both Q-PCR and Western blot detection. Next, to examine the roles of Frizzled-2in Wnt5a signaling and calcium inflow, we transfected rat H9c2cells with synthesized frizzled-2plasmid and then analyzed the expression of Frizzle-2and Wnt5a collected from Q-PCR and Western blot. When the cells were transfected, we observed that gene expressions of both frizzled-2and Wnt5a were significantly increased approximately12-fold and10-fold, respectively, in comparison to that in non-transfected cells (P<0.001). Moreover, protein expressions of both Frizzled-2and Wnt5a were significantly increased approximately8-and3-fold, respectively, when compared with that in non-transfected cells (P<0.001). In addition, it is known that phosphorylation of CaMK II indicates the action of the frizzled-2mediate Wnt5a signaling pathway [35], and we found that p-CaMK Ⅱ was also significantly increased4-fold after the transfection (P<0.001;), compared to non-transfected cells.To identify whether activation of the Wnt5a/Frizzled-2pathway regulates Ca2+ release from intracellular stores, we examined whether changes in intracellular Ca2+inflow was affected by frizzled-2-transfection, indicated by the intensity of fluorescence of fluo-3/AM under confocal microscope after transfection. We found that the intensity was significantly increased2.5-fold after the transfection (P<0.001) when compared to the normal cells. This indicated that intracellular Ca2+inflow was increased in frizzled-2-transfected H9c2cells.
As described above, we showed that the expression of Wnt5a was closely associated with the expression of the Frizzled-2receptor, and that cells transfected with frizzled-2plasmid dramatically increased intracellular Ca2+inflow. Nevertheless, we could not draw a conclusion that Frizzled-2transduces binding of Wnt5a and leads to the intracellular Ca2+release. To resolve this problem, a special inhibitor called stealth RNAi was used to block the expression of the frizzled-2gene. Compared to the treatment without stealth RNAi, we found that the treatment with stealth RNAi significantly inhibited both gene and protein expression of Frizzled-2in transfected H9c2cells nearly to the control level, as demonstrated by Q-PCR and Western blot (P<0.001). Moreover, the expressions of the Wnt5a gene and protein were also markedly downregulated in transfected H9c2cells following the suppression of the frizzled-2gene by the treatment of stealth RNAi (P<0.001), compared to that without this treatment. To examine whether the expression of p-CaMKⅡ was blocked by the inhibition of frizzled-2signaling, we detected its expression by Western blot after application of stealth RNAi. We found that the treatment of stealth RNAi significantly inhibited the expression of p-caMK Ⅱ by1.5-fold in frizzled-2transfected H9c2cells, indicating that the activation of the Wnt5a/Frizzled-2pathway was suppressed by blocking frizzled-2.
To examine whether intracellular Ca2+inflow was affected by blocking frizzled-2signaling, we measured the intensity of fluorescence of fluo-3/AM under confocal microscope after application of stealth RNAi. We found that stealth RNAi dramatically decreased the intensity of fluorescence in frizzled-2transfected H9c2cells, compared to that without the treatment. Taken together, we can draw a conclusion that the Wnt5a/Frizzled-2pathway exists in rat H9c2cells and activates Ca2+release from intracellular stores.
Part two:Ca2+overload is known to be an important mechanism after I/R. In the adult heart and blood vessels, Wnt signaling activity is quite low under normal conditions. However, this pathway is reactivated during the pathological cardiac remodeling induced by pressure overload, in injured arteries and after myocardial infarction. In an initial study by Blankesteijn, et al, frizzled-2mRNA levels were found to be gradually upregulated in the first10days after myocardial infarction in the rat. Therefore, we used a rat I/R model to test whether the Wnt5a/Frizzled-2signaling pathway relative elements upregulate in injured cadiocyte. Firstly, we detected the expression of Frizzled-2and Wnt5a in normal rat H9c2cells on both the gene and protein level. We found that gene and protein expressions of both Frizzled-2and Wnt5a were expressed in normal cadiocyte. However, after I/R, gene expressions of frizzled-2and Wnt5a were significantly elevated approximately8-and3-fold, respectively, in the cadiocyte, when compared with that in normal H9c2cells (P<0.001and P<0.05, respectively). Protein expressions of Frizzled-2and Wnt5a were also elevated by4-and6-fold in cadiocyte, respectively, when compared to that in normal cadiocyte (P<0.001).While p-CaMKII was expressed at a very low level in normal cadiocyte, however, its expression was markedly increased approximately7-fold in the cadiocyte after I/R (P<0.001).
Because the mechanism of intracellular Ca2+accumulation after I/R are complicate, and hard to examine whether Wnt5a/Frizzled-2pathway contributes to the Ca2+accumulation after in vivo. So we choose to verify our hypothsis in pure populations of these cells.
Part three:we used H/R to mimic I/R in vivo [35,36]. Following H/R, We analyzed the expression of Frizzle-2and Wnt5a collected from Q-PCR and Western blot, observed that gene expressions of both frizzled-2and Wnt5a were significantly increased approximately8-fold and3-fold, respectively, in comparison to that normal cells (P<0.001). Moreover, protein expressions of both Frizzled-2and Wnt5a were significantly increased approximately7-and2-fold, respectively, when compared with that in normal cells (P<0.001).We found that the Ca2+intensity was significantly increased2-fold after H/R (P<0.001) when compared to nomal cells. This indicated that intracellular Ca2+inflow was increased in H/R treatment H9c2cells.In addition, we found that p-CaMK II was also significantly increased4-fold after the H/R (P<0.001), compared to non-transfected cells.
To determine the interaction between H/R-induced activation of Wnt5a/Frizzled-2signaling and accumulation of intracellular Ca2+, we suppressed the expression of the frizzled-2receptor gene by transfect with stealth RNAi before H/R, and detected the effects of stealth RNAi on gene and protein expression of Frizzled-2and Wnt5a after H/R. we observed a significant inhibitory effect on gene expression of frizzled-2in the transfected H9c2cells, compared with that in cells without transfect with stealth RNAi, as demonstrated by the fact that gene expression of frizzled-2was inhibited nearly6-fold. Surprisingly, the expression of the Wnt5a gene was also downregulated nearly3-fold (P<0.001). In addition, protein expressions of Frizzled-2and Wnt5a in transfected cells was significant decreased when compared to cells without transfection (P<0.001). Encouraging, the intensity of Ca2+fluorescence was significantly decreased in transfected cells after the H/R, when compared to those without transfection(P<0.001).Moreover, p-CaMKII in transfected cells was also inhibited by nearly3-fold, compare to only H/R treatment cells (P<0.001).
Conclusion and Significance
According to the results from this study we can draw the following conclusions:
(1) Wnt5a/Frizzled-2signaling pathways exist in mammals myocardial cells;
(2) Expression of Frizzled-2and Wnt5a were upregulated after myocardial ischemia reperfusion injury.
(3) We used hypoxia/reoxygenation to mimic myocardial ischemia-reperfusion injury in H9c2cells can activate Wnt5a/Frizzled-2signaling pathway. And found out that this pathway contribute to calcium overload after hypoxia/reoxygenation. In other words, Wnt5a/Frizzled-2signaling pathway was activated after hypoxia/reoxygenation, may may be one of the mechanisms of calcium overload after myocardial ischemia-reperfusion injury.
(3) This study tested the reliability of Stealth RNAi gene silencing, it has high inhibition efficiency;
By research on Wnt5a/Frizzled-2signaling pathways in myocardial ischemia reperfusion injury, may contribute to understanding the futher mechanism of reperfusion injury, will provide a new potential therapeutic target for treatment and research.
引文
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