cGMP的心肌保护作用及自噬在MIRI中的作用
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摘要
第一部分
环磷酸鸟苷(cyclic guanosine 3'5'-monophosphate, cGMP)是细胞内一种第二信使,是由可溶性鸟苷酸环化酶(soluble guanosine cyclase, sGC)和颗粒状鸟苷酸环化酶(particulate guanylyl cyclase, pGC)催化GTP转化而生成。作为一种重要的细胞内信使物质,cGMP有3个主要靶点：cGMP-依赖的蛋白激酶或蛋白激酶G (protein kinase G, PKG), cGMP-调节的cAMP磷酸二酯酶,以及cGMP控制的离子通道。其中,PKG被认为是最重要的cGMP下游靶点。在生理条件下,PKG通过松弛平滑肌细胞、抑制内皮细胞通透性、抑制血小板活化以及对心肌细胞的负性肌力作用保护心血管系统。最近有研究报道,cGMP/PKG信号途径在缺血预处理和后处理的心脏保护机制中发挥重要作用。另有报道指出,磷酸二酯酶-5抑制剂西地那非(sildenafil,伟哥)通过增加cGMP的积聚激活PKG,进而保护缺血／再灌注的心肌细胞。然而cGMP/PKG信号途径保护心脏的具体机制尚不清楚。
目前研究认为,线粒体膜通透性转移孔(mitochondrial permeability transition pore, mPTP)的开放是导致缺血／再灌注损伤的关键因素,抑制mPTP的开放是许多心脏保护物质抗缺血／再灌注损伤的共同机制。虽然有人提出,mPTP可能是cGMP/PKG信号途径引起心脏保护的最终靶点,但尚未被证实。糖原合成酶激酶3β(glycogen synthase kinase 3p, GSK-3p)在心肌保护中也起非常重要的作用,其机制主要是通过Ser9位点的磷酸化抑制其活性,从而阻止mPTP的开放并保护再灌注损伤的心肌。已有研究证明,西地那非通过PKG信号通路使心肌细胞GSK-3β失活。推测,cGMP很可能通过GSK-3p的失活抑制mPTP的开放。磷脂酰肌醇三激酶(phosphoinositide 3-kinase, PI3K)／蛋白激酶B (Akt)信号途径抑制GSK-3p的活性,并有报道此途径可能被西地那非激活。同样可以设想,cGMP/PKG信号可能通过PI3K/Akt使GSK-3β失活。PI3K/Akt的激活虽对缺血预处理心脏起保护作用,但是过度的Akt活化可以诱发病理性心室重构和心力衰竭。因此,探讨cGMP对Akt活性的影响及其可能机制具有重要意义。
本实验利用H9c2大鼠心肌细胞株,探讨cGMP的心脏保护作用是否通过GSK-3β的失活并抑制mPTP开放而引起,并进一步观察cGMP是否通过影响Akt的活性使GSK-3p失活。
为了探讨cGMP能否使GSK-3β失活,本研究用cGMP类似物8-Br-cGMP (500μmol/L)处理H9c2细胞30 min,利用Western blotting方法观察GSK-3βSer9位点磷酸化的情况。结果显示,8-Br-cGMP明显增加GSK-3p的磷酸化,表明cGMP能够抑制GSK-3β的活性。因为PKG是cGMP的重要下游因子,本实验通过测定PKG的主要底物血管扩张刺激磷蛋白(vasodilator-stimulated phosphoprotein, VASP)的磷酸化水平检测了PKG的活性。结果显示,8-Br-cGMP明显增加VASP的磷酸化,提示8-Br-cGMP能够激活PKG。cGMP抑制GSK-3p活性的效应被特异性PKG抑制剂KT5823 (10μmol/l)所逆转,提示cGMP是通过PKG抑制GSK-3β的活性。进一步的实验显示,8-Br-cGMP本身并不能明显增强GSK-3β磷酸化,而必须和PKG la共同作用时才表现出增加GSK-3p磷酸化的作用,进一步证实了cGMP通过PKG抑制GSK-3β的活性。同样,8-Br-cGMP与PKG la的直接相互作用使纯化GSK-3p的磷酸化明显增加。以上结果表明cGMP通过PKG使GSK-3p失活。
为了探讨cGMP能否阻止mPTP开放,本实验利用激光共聚焦显微镜成像技术和荧光染色方法(四甲基罗丹明乙酯tetramethyrhodamine ester, TMRE)测定了线粒体膜电位,以此判断mPTP的开放程度。结果显示,cGMP明显抑制氧化应激诱导的TMRE荧光强度的减少,说明cGMP能够抑制mPTP的开放。用激活型GSK-3β质粒(GSK-3β-S9A)转染细胞,使GSK-3p处于持续激活状态后,cGMP没能抑制TMRE荧光强度的减少。由于抑制GSK-3β的活性可阻止mPTP的开放,此结果进一步证明cGMP通过抑制GSK-3β活性而阻止mPTP的开放。
为了进一步阐明cGMP是否通过影响Akt活性而使GSK-3β失活,实验用Western blotting检测了Akt Ser473位点的磷酸化。我们意外地发现,8-Br-cGMP不能增加Akt的磷酸化,反而使其减少,提示cGMP的积聚可迅速抑制Akt的活性。为了证实这一发现,我们进一步检测了非特异性磷酸二酯酶抑制剂异丁基甲基黄嘌呤(3-isobutyl-1-methylxanthine, IBMX,200μmol/L)能否模拟cGMP的这种作用而抑制Akt的磷酸化。结果表明,IBMX使Akt的磷酸化明显减少,却增强VASP的磷酸化,表明IBMX通过cGMP抑制Akt的活性。进一步的实验结果显示,8-Br-cGMP也阻断胰岛素对Akt的磷酸化效应,提示cGMP不仅抑制Akt基础活性,也抑制配体诱发的Akt活化。因为Akt的过度激活可导致心肌肥大和心力衰竭,本实验结果可能提示,cGMP可防止心衰的发生。本实验中还发现cGMP对Akt磷酸化的抑制作用不能被PKG的抑制剂KT5823所逆转,并且PKG的RNA敲除亦不能影响cGMP对Akt磷酸化的抑制效应,说明虽然PKG作为cGMP的重要下游信号,通过使GSK-3p失活而阻止mPTP的开放,但并不参与cGMP对Akt的负性调节作用。
Thr308和Ser473位点的磷酸化使Akt活化,而丝／苏氨酸蛋白激酶磷酸酶使这些残基去磷酸化而导致Akt的失活。因此,我们探讨了cGMP是否通过激活丝／苏氨酸蛋白激酶磷酸酶而使Akt失活。结果显示,丝／苏氨酸蛋白激酶磷酸酶抑制剂Calyculin A (5 nmol/L)使Akt Ser473位点磷酸化明显增加,且此效应被8-Br-cGMP所阻断,提示cGMP能活化丝／苏氨酸蛋白激酶磷酸酶,从而导致Akt失活。为了进一步证实这一发现,我们利用比色法进一步检测了蛋白磷酸酶2A(protein phosphatase 2A, PP2A)的活性。结果显示,8-Br-cGMP使PP2A活性明显增加。这些结果均表明,cGMP通过激活蛋白激酶磷酸酶尤其是PP2A而抑制Akt的活性。
小结
1. cGMP通过PKG抑制GSK-3β的活性并阻止mPTP的开放,从而保护心脏；
2. cGMP对Akt活性起负性调节作用；
3. cGMP抑制Akt的活性主要通过激活PP2A,而不是通过PKG信号通路；
4. cGMP对心肌的生存具有双重调节作用。
第二部分
缺血性心脏病(ischemic heart disease, IHD)是严重威胁人类健康的最常见疾病之一。IHD的基本病理生理过程是心肌缺血,在其治疗过程中再灌注会引发缺血区功能障碍加重和结构损伤,称为再灌注损伤(reperfusion injury)。有效防治再灌注损伤己成为医学界亟待解决的重要课题。再灌注损伤的发生机制十分复杂,且尚未完全阐明。普遍认为,自由基、钙超载、心肌能量代谢障碍、炎性反应与细胞凋亡等在再灌注损伤中起重要作用。自噬(Autophagy)作用是广泛存在于大部分真核细胞中的一种生命现象,是溶酶体对自身结构的吞噬降解,它是细胞内的再循环系统。细胞在基础状态下有自噬活动,同时各种应激如饥饿和低氧症也可以诱发自噬。自噬是一种细胞生存机制,但在某些条件下也导致细胞死亡(自噬性死亡或Ⅱ型程序性死亡)。最近的研究表明,心肌缺血／再灌注时可诱发自噬,但其发生的时期和具体作用尚不清楚。弄清缺血／再灌注过程中自噬启动时期及强度的演变,对再灌注损伤的防治可能起重要的指导作用。自噬的调控主要是由Ⅰ型和Ⅲ型磷酸肌醇三磷酸激酶(PI3K)来完成,其中Ⅰ型PI3K通过激活雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)来抑制自噬的发生,即负性调节机制；自噬的另一种调节方式为mTOR非依赖性调节机制,也称为正性调节机制,是Ⅲ型PI3K通过增加Beclin 1蛋白的表达而诱发。LC3是Atg8在哺乳动物中的同源物,已被明确参与自噬过程。LC3有LC3-I、LC3-II两种亚型,其中LC3-Ⅰ是胞浆蛋白,LC3-Ⅱ定位于前自噬体和自噬体,LC3-Ⅱ／LC3-Ⅰ比值是自噬体形成数量的重要标志。了解心肌缺血／再灌注时自噬发生的调控机制,有助于认识再灌注损伤的发病机制,并有助于寻找治疗再灌注损伤的有效方法。基于以上理论,本研究初步探讨了心肌缺血／再灌注时自噬的发生时期、自噬在再灌注损伤中的具体作用以及其调控机制。
本实验首先用Langendorff装置制备大鼠离体心脏缺血／再灌注损伤模型,在缺血0、10、20和30 min时和再灌注10、30、60和120 min时采集心脏组织标本,用Western blotting方法检测LC3蛋白的表达。结果显示,在缺血期LC3-Ⅱ／LC3-Ⅰ比值虽有增加趋势,但与对照组相比差异无统计学意义,自噬活动并没有加强。而在再灌注期不同时间点自噬活动均显著加强,说明自噬在心肌再灌注损伤中起一定作用。
为了进一步探讨自噬在再灌注损伤中的确切作用,本研究观察了3-甲基腺嘌呤(3-methyladenine,3-MA)和腺苷受体激动剂(N-Ethylcarboxamidoadenosine, NECA)对自噬和心肌梗死的影响。3-MA为特异性自噬抑制剂,而NECA则为腺苷A2受体拟似剂,是公认的心脏保护物质。再灌注5 min之前开始用3-MA和NECA进行干预并持续35 min。于再灌注10、30、60和120 min时采集心脏组织标本,用Western blotting检测LC3蛋白的表达,以观察3-MA和NECA对自噬的影响。再灌注120 min结束后,TTC染色法测定心肌梗死面积以观察两种干预药物对心肌梗死面积大小的影响,推测自噬在再灌注损伤中的具体作用。结果显示,3-MA抑制再灌注损伤诱发的自噬,并减少心肌梗死面积,说明自噬在心肌再灌注损伤中起有害作用。NECA既能抑制自噬,同时也减轻再灌注引起的心肌梗死。这说明NECA抑制自噬活动可能是其保护再灌注心脏的机制之一。
为了探讨再灌注诱发自噬的机制,本研究用Western blotting方法分别检测了缺血期和再灌注期Beclin 1和mTOR的活性。结果显示,在缺血期和再灌注期,Beclin 1蛋白的表达均没有增加,反而出现了下降的趋势。Beclin 1是自噬正性调节机制中的关键基因,说明在大鼠离体心脏缺血／再灌注损伤模型中,自噬的发生并不主要依赖于其正性调节机制。进一步实验显示,mTOR的活性在缺血期和再灌注期均降低,说明负性调节机制的减弱可能是诱发自噬的原因。
小结
1.在大鼠离体心脏缺血／再灌注损伤模型中,自噬在再灌注期发生；
2.自噬活动在大鼠离体心脏再灌注损伤中起有害作用；
3.在大鼠离体心脏,再灌注诱发的自噬主要是通过负性调节机制引起,其正性调节机制不起主要作用。
Part1
Cyclic guanosine 3'5'-monophosphate (cGMP) is a second messenger molecule and is generated from the catalytic conversion of guanosine triphosphate (GTP) by the soluble guanylyl cyclase (sGC) and the particulate guanylyl cyclase (pGC) As an important cellular messenger, cGMP has three major identified targets: cGMP-dependent protein kinase or protein kinase G (PKG), cGMP-regulated cAMP phosphodiesterase, and cGMP-gated ion channels. Among these targets, PKG has been identified as the most important downstream signal of cGMP. Under physiological conditions, PKG exerts a beneficial influence on the cardiovascular system through relaxation of smooth muscle cells, inhibition of endothelial permeability, inhibition of platelet activation, and negative inotropic effect on cardiomyocytes. Recent studies have shown that the cGMP/PKG signaling pathway is involved in the cardioprotective mechanism underlying ischemic preconditioning and postconditioning. It has also been reported that inhibition of phosphodiesterase-5 with sildenafil (Viagra) which can augment cGMP accumulation protects cardiomyocytes from ischemia/reperfusion injury via a PKG-dependent mechanism. However, the mechanism underlying the cardioprotective effects mediated by the cGMP/PKG signaling pathway remains to be uncertain.
Opening of the mPTP contributes to the pathogenesis of ischemia/reperfusion injury, whereas modulation of the mPTP opening has been proposed to be the common mechanism by which various cardioprotective interventions confer protection against ischemia/reperfusion injury. Although the mPTP has been proposed to be the target of the cGMP/PKG signaling, the impact of the cGMP/PKG on the pore opening is unknown. Studies have shown that glycogen synthase kinase 3β(GSK-3β) may play such a role by interacting with the mPTP after being inactivated by the upstream protective signals. Since sildenafil inactivates GSK-3βvia PKG in cardiomyocytes, it is likely that cGMP may lead to prevention of the mPTP opening by inactivating GSK-3β. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is well known for its negative effect on GSK-3βactivity, and is reported to be activated by sildenafil. Thus, it is possible that the cGMP/PKG signaling inactivates GSK-3βthrough PI3K/Akt. Although Akt activation contributes to cardioprotection of ischemic preconditioning, an excessive activation of Akt also leads to pathologic remodeling and heart failure. Thus, it is intriguing to address the mechanism responsible for the inhibitory effect of cGMP on Akt activity.
The purpose of this study was to determine if cGMP prevents the mPTP opening by inactivating GSK-3(3 in cardiac H9c2 cells and to test if cGMP inactivates GSK-3βby altering Akt activity. To test if cGMP could inactivate GSK-3β, we treated H9c2 cells with the cGMP analogue 8-Br-cGMP (500μmol/L) for 30 min and detected GSK-3P phosphorylation at Ser9 with Western blot. Our data showed that 8-Br-cGMP increased GSK-3βphosphorylation in a dose-dependent manner, indicating cGMP can inactivate GSK-3β. Since PKG is the most important downstream signal of cGMP, we examined the potential role of PKG in the action of 8-Br-cGMP by detecting the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP), a major substrate of PKG. The effect of cGMP on GSK-3βactivity was reversed by the selective PKG inhibitor KT5823 (10μmol/L), indicating that PKG accounts for the action of cGMP. Experiments with cell lysates showed that 8-Br-cGMP alone did not alter but together with PKG la markedly enhanced GSK-3βphosphorylation, confirming that cGMP inhibits GSK-3βthrough PKG. In support, in vitro experiments revealed that interaction of 8-Br-cGMP with PKG la markedly enhanced phosphorylation of purified GSK-3β. This result also indicates that GSK-3βis a direct substrate of PKG. These results clearly demonstrate that cGMP inactivates GSK-3βvia PKG. To determine if cGMP can prevent the mPTP opening, we examined the effect of 8-Br-cGMP on oxidant-induced loss ofΔΨm by monitoring changes in TMRE fluorescence with confocal microscopy. cGMP prevented the loss of TMRE fluorescence caused by H2O2, indicating that cGMP can suppress the mPTP opening. cGMP failed to preserve TMRE fluorescence in cells transfected with the constitutively active GSK-3β(GSK-3P-S9A) mutant plasmid, suggesting that cGMP modulates the mPTP opening by inactivating GSK-3β.
To test if Akt activation contributes to the inhibitory effect of cGMP on GSK-3βactivity, we measured Akt phosphorylation at Ser473 with Western blot. To our great surprise,8-Br-cGMP did not increase but decreased Akt phosphorylation under physiological conditions, indicating that an accumulation of cGMP rapidly inactivates Akt. To confirm this finding, we further tested if the non-specific inhibitor of phosphodiesterases isobutylmethylxanthine (IBMX,200μmol/L) which maintains intracellular cGMP accumulation by inhibiting enzymatic hydrolysis of cGMP, could also mimic the effect of cGMP to inhibit Akt phosphorylation. IB MX markedly reduced Akt phosphorylation but enhanced VASP phosphorylation, indicating that IBMX indeed negatively regulates Akt activity via cGMP. Further experiments showed that 8-Br-cGMP suppresses the effect of insulin on Akt phosphorylation, suggesting that cGMP not only suppresses the basal Akt activity but also impedes the ligand-induced Akt activation. Because an excessive activation of Akt may lead to cardiac hypertrophy and heart failure, our finding suggests that cGMP may prevent hypertrophy and heart failure. The inhibitory effect of cGMP on Akt phosphorylation was not reversed by both the PKG inhibitor KT5823 and PKG siRNA, suggesting that PKG is not required for the negative regulatory action of cGMP on Akt signaling, although it serves as the main downstream signal of cGMP to prevent the mPTP opening by inactivating GSK-3β.
Akt is activated by phosphorylation of Thr308 and Ser473, and dephosphorylation of these residues by Ser/Thr protein phosphatases inactivates the kinase. Thus, we tested if cGMP could inactivate Akt by activating Ser/Thr protein phosphatases. Calyculin A (5 nmol/L), a cell permeable Ser/Thr protein phosphatase inhibitor, dramatically increased Akt phosphorylation, an effect that was partially but significantly blocked by 8-Br-cGMP, indicating that cGMP is able to activate Ser/Thr protein phosphatases, which may lead to inactivation of Akt. To corroborate this finding, we further detected the activity of protein phosphatase 2A (PP2A), a major Ser/Thr protein phosphatase.8-Br-cGMP significantly increased PP2A activity, indicating that cGMP may inhibit Akt activity by activating PP2A.
Summary
1. Cyclic GMP prevents the mPTP opening by inactivating GSK-3βvia PKG in H9c2 cells, which accounts for the cardioprotective of cGMP;
2. Cyclic GMP negatively regulates Akt activity;
3. Cyclic GMP inactivates Akt through activation of PP2A but not through PKG;
4.Cyclkic GMP has duel roles in cardiac survival.
Part1
Ischemic heart disease (IHD) is the leading cause of morbidity and mortality worldwide. The fundamental cause of IHD is myocardial ischemia. Reperfusion therapies cause myocardial contractile dysfunction and tissue damage in the ischemic zone, so-called myocardial reperfusion injury. Development of effective therapies to protect reperfusion injury is an important issue of the medical community. The mechanism underlying myocardial reperfusion injury is complicated and remains unclear, although free radicals, calcium overload, defects in energy metabolism, inflammation, and apoptosis have been proposed to account for the injury. Recent studies indicate that autophagy may play a role in myocardial reperfusion injury. Autophagy occurs constitutively in eukaryotic cells and is a process involving the degradation of cell's own components through the lysosomal machinery. Autophagy occurs under normal conditions, but is also upregulated in response to stress such as starving and hypoxia. Although autophagy can promote cell survival, it may also cause cell death (autophagic cell death or type II programmed cell death). While studies have shown that ischemia/reperfusion can trigger autophagy, the exact induction timing and role of autophagy are unclear. Understanding of the detailed time course for the induction and progression of autophagy during ischemia/reperfusion is critical for the development of preventive and therapeutic interventions for reperfusion injury. Autophagy is regulated by the class I and III of PI3K. The class I inhibits autophagy through mammalian target of rapamycin (mTOR) (the negative pathway), whereas the class III PI3K activates autophagy by increasing Beclin-1 expression, so-called the positive pathway or non mTOR-dependent pathway. LC3, the rat microtubule-associated protein 1 light chain 3, a mammalian homologue of yeast ATG8, is associated with autophagy. There are two forms of LC3, namely, LC3-Ⅰ(cytosol form) and LC3-Ⅱ(processed form). The ratio of LC3-Ⅱ/LC3-Ⅰis correlated with the extent of autophagosome formation and thus was suggested as an excellent marker of autophagy. Investigation of the regulatory mechanism of autophagy occurring during reperfusion is important for understanding of the mechanism underlying reperfusion injury and will help discover effective therapeutic intervention to prevent reperfusion injury. Based on the above theoretical interpretations, the current study investigated the time course of autophagy induction, the exact role of autophagy during reperfusion injury, and the regulatory mechanism underlying the induction of autophagy.
Isolated rat hearts were perfused on a Langendorff apparatus and subjected to 30 min regional ischemia followed by 2 h of reperfusion. Biopsies were collected from the risk zones during ischemia (0,10,20 and 30 min of ischemia) and reperfusion (10,30,60, and 120 min after the onset of reperfusion), and autophagy was determined by the ratio of LC3-Ⅱto LC3-Ⅰwith Western blotting. We found that although LC3-Ⅱ/LC3-Ⅰratio had a tendency to increase during ischemia, there was no statistical difference when compared to the sham group, indicating that autophagy is not prominent during ischemia. In contrast, LC3-Ⅱ/LC3-Ⅰratio was markedly increased upon reperfusion, implying that autophagy may play a role in reperfusion injury.
Since autophagy may play a role during reperfusion, we treated rat hearts with 3-MA and NEC A starting 5 min before the onset of reperfusion for 35 min. Myocardial samples were collected form the risk zones 10,30,60, and 120 min after the onset of reperfusion. Autophagy was determined by the ratio of LC3-Ⅱto LC3-Ⅰwith Western blotting, whereas myocardial infarction was measured with TTC staining. Our data showed that 3-MA not only inhibited autophagy during reperfusion but also reduced infarct size, suggesting that autophagy plays a detrimental role in reperfusion injury. Similarly, NEC A also reduced both autophagy and myocardial infarction, indicating that inhibition of autophagy may contribute to the protective effect of NEC A on reperfusion injury.
To investigate the mechanism underlying the induction of autophagy, we detected Beclin 1 expression and mTOR activities during ischemia and reperfusion with Western blotting. Either ischemia or reperfusion did not enhance Beclin 1 expression, suggesting Beclin 1 may not be critical for the formation of autophagy. Since Beclin 1 is a critical gene for the positive pathway, our observation suggests that the induction of autophagy during reperfusion is not dependent on the positive pathway in isolated rat hearts. In contrast, the activity of mTOR was decreased during both ischemia and reperfusion, indicating that an inhibition of the negative pathway may account for autophagy in the setting of ischemia/reperfusion.
Summary
1. Autophagy is induced upon reperfusion but not during ischemia in isolated rat hearts;
2. Autophagy plays a detrimental role during ischemia/reperfusion;
3. Inhibition of the negative pathway but not the positive pathway account for the formation of autophagy in isolated rat hearts.
环磷酸鸟苷(cyclic guanosine 3'5'-monophosphate, cGMP)是细胞内一种第二信使,是由可溶性鸟苷酸环化酶(soluble guanosine cyclase, sGC)和颗粒状鸟苷酸环化酶(particulate guanylyl cyclase, pGC)催化GTP转化而生成。作为一种重要的细胞内信使物质,cGMP有3个主要靶点：cGMP-依赖的蛋白激酶或蛋白激酶G (protein kinase G, PKG), cGMP-调节的cAMP磷酸二酯酶,以及cGMP控制的离子通道。其中,PKG被认为是最重要的cGMP下游靶点。在生理条件下,PKG通过松弛平滑肌细胞、抑制内皮细胞通透性、抑制血小板活化以及对心肌细胞的负性肌力作用保护心血管系统。最近有研究报道,cGMP/PKG信号途径在缺血预处理和后处理的心脏保护机制中发挥重要作用。另有报道指出,磷酸二酯酶-5抑制剂西地那非(sildenafil,伟哥)通过增加cGMP的积聚激活PKG,进而保护缺血／再灌注的心肌细胞。然而cGMP/PKG信号途径保护心脏的具体机制尚不清楚。
目前研究认为,线粒体膜通透性转移孔(mitochondrial permeability transition pore, mPTP)的开放是导致缺血／再灌注损伤的关键因素,抑制mPTP的开放是许多心脏保护物质抗缺血／再灌注损伤的共同机制。虽然有人提出,mPTP可能是cGMP/PKG信号途径引起心脏保护的最终靶点,但尚未被证实。糖原合成酶激酶3β(glycogen synthase kinase 3p, GSK-3p)在心肌保护中也起非常重要的作用,其机制主要是通过Ser9位点的磷酸化抑制其活性,从而阻止mPTP的开放并保护再灌注损伤的心肌。已有研究证明,西地那非通过PKG信号通路使心肌细胞GSK-3β失活。推测,cGMP很可能通过GSK-3p的失活抑制mPTP的开放。磷脂酰肌醇三激酶(phosphoinositide 3-kinase, PI3K)／蛋白激酶B (Akt)信号途径抑制GSK-3p的活性,并有报道此途径可能被西地那非激活。同样可以设想,cGMP/PKG信号可能通过PI3K/Akt使GSK-3β失活。PI3K/Akt的激活虽对缺血预处理心脏起保护作用,但是过度的Akt活化可以诱发病理性心室重构和心力衰竭。因此,探讨cGMP对Akt活性的影响及其可能机制具有重要意义。
本实验利用H9c2大鼠心肌细胞株,探讨cGMP的心脏保护作用是否通过GSK-3β的失活并抑制mPTP开放而引起,并进一步观察cGMP是否通过影响Akt的活性使GSK-3p失活。
为了探讨cGMP能否使GSK-3β失活,本研究用cGMP类似物8-Br-cGMP (500μmol/L)处理H9c2细胞30 min,利用Western blotting方法观察GSK-3βSer9位点磷酸化的情况。结果显示,8-Br-cGMP明显增加GSK-3p的磷酸化,表明cGMP能够抑制GSK-3β的活性。因为PKG是cGMP的重要下游因子,本实验通过测定PKG的主要底物血管扩张刺激磷蛋白(vasodilator-stimulated phosphoprotein, VASP)的磷酸化水平检测了PKG的活性。结果显示,8-Br-cGMP明显增加VASP的磷酸化,提示8-Br-cGMP能够激活PKG。cGMP抑制GSK-3p活性的效应被特异性PKG抑制剂KT5823 (10μmol/l)所逆转,提示cGMP是通过PKG抑制GSK-3β的活性。进一步的实验显示,8-Br-cGMP本身并不能明显增强GSK-3β磷酸化,而必须和PKG la共同作用时才表现出增加GSK-3p磷酸化的作用,进一步证实了cGMP通过PKG抑制GSK-3β的活性。同样,8-Br-cGMP与PKG la的直接相互作用使纯化GSK-3p的磷酸化明显增加。以上结果表明cGMP通过PKG使GSK-3p失活。
为了探讨cGMP能否阻止mPTP开放,本实验利用激光共聚焦显微镜成像技术和荧光染色方法(四甲基罗丹明乙酯tetramethyrhodamine ester, TMRE)测定了线粒体膜电位,以此判断mPTP的开放程度。结果显示,cGMP明显抑制氧化应激诱导的TMRE荧光强度的减少,说明cGMP能够抑制mPTP的开放。用激活型GSK-3β质粒(GSK-3β-S9A)转染细胞,使GSK-3p处于持续激活状态后,cGMP没能抑制TMRE荧光强度的减少。由于抑制GSK-3β的活性可阻止mPTP的开放,此结果进一步证明cGMP通过抑制GSK-3β活性而阻止mPTP的开放。
为了进一步阐明cGMP是否通过影响Akt活性而使GSK-3β失活,实验用Western blotting检测了Akt Ser473位点的磷酸化。我们意外地发现,8-Br-cGMP不能增加Akt的磷酸化,反而使其减少,提示cGMP的积聚可迅速抑制Akt的活性。为了证实这一发现,我们进一步检测了非特异性磷酸二酯酶抑制剂异丁基甲基黄嘌呤(3-isobutyl-1-methylxanthine, IBMX,200μmol/L)能否模拟cGMP的这种作用而抑制Akt的磷酸化。结果表明,IBMX使Akt的磷酸化明显减少,却增强VASP的磷酸化,表明IBMX通过cGMP抑制Akt的活性。进一步的实验结果显示,8-Br-cGMP也阻断胰岛素对Akt的磷酸化效应,提示cGMP不仅抑制Akt基础活性,也抑制配体诱发的Akt活化。因为Akt的过度激活可导致心肌肥大和心力衰竭,本实验结果可能提示,cGMP可防止心衰的发生。本实验中还发现cGMP对Akt磷酸化的抑制作用不能被PKG的抑制剂KT5823所逆转,并且PKG的RNA敲除亦不能影响cGMP对Akt磷酸化的抑制效应,说明虽然PKG作为cGMP的重要下游信号,通过使GSK-3p失活而阻止mPTP的开放,但并不参与cGMP对Akt的负性调节作用。
Thr308和Ser473位点的磷酸化使Akt活化,而丝／苏氨酸蛋白激酶磷酸酶使这些残基去磷酸化而导致Akt的失活。因此,我们探讨了cGMP是否通过激活丝／苏氨酸蛋白激酶磷酸酶而使Akt失活。结果显示,丝／苏氨酸蛋白激酶磷酸酶抑制剂Calyculin A (5 nmol/L)使Akt Ser473位点磷酸化明显增加,且此效应被8-Br-cGMP所阻断,提示cGMP能活化丝／苏氨酸蛋白激酶磷酸酶,从而导致Akt失活。为了进一步证实这一发现,我们利用比色法进一步检测了蛋白磷酸酶2A(protein phosphatase 2A, PP2A)的活性。结果显示,8-Br-cGMP使PP2A活性明显增加。这些结果均表明,cGMP通过激活蛋白激酶磷酸酶尤其是PP2A而抑制Akt的活性。
小结
1. cGMP通过PKG抑制GSK-3β的活性并阻止mPTP的开放,从而保护心脏；
2. cGMP对Akt活性起负性调节作用；
3. cGMP抑制Akt的活性主要通过激活PP2A,而不是通过PKG信号通路；
4. cGMP对心肌的生存具有双重调节作用。
第二部分
缺血性心脏病(ischemic heart disease, IHD)是严重威胁人类健康的最常见疾病之一。IHD的基本病理生理过程是心肌缺血,在其治疗过程中再灌注会引发缺血区功能障碍加重和结构损伤,称为再灌注损伤(reperfusion injury)。有效防治再灌注损伤己成为医学界亟待解决的重要课题。再灌注损伤的发生机制十分复杂,且尚未完全阐明。普遍认为,自由基、钙超载、心肌能量代谢障碍、炎性反应与细胞凋亡等在再灌注损伤中起重要作用。自噬(Autophagy)作用是广泛存在于大部分真核细胞中的一种生命现象,是溶酶体对自身结构的吞噬降解,它是细胞内的再循环系统。细胞在基础状态下有自噬活动,同时各种应激如饥饿和低氧症也可以诱发自噬。自噬是一种细胞生存机制,但在某些条件下也导致细胞死亡(自噬性死亡或Ⅱ型程序性死亡)。最近的研究表明,心肌缺血／再灌注时可诱发自噬,但其发生的时期和具体作用尚不清楚。弄清缺血／再灌注过程中自噬启动时期及强度的演变,对再灌注损伤的防治可能起重要的指导作用。自噬的调控主要是由Ⅰ型和Ⅲ型磷酸肌醇三磷酸激酶(PI3K)来完成,其中Ⅰ型PI3K通过激活雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)来抑制自噬的发生,即负性调节机制；自噬的另一种调节方式为mTOR非依赖性调节机制,也称为正性调节机制,是Ⅲ型PI3K通过增加Beclin 1蛋白的表达而诱发。LC3是Atg8在哺乳动物中的同源物,已被明确参与自噬过程。LC3有LC3-I、LC3-II两种亚型,其中LC3-Ⅰ是胞浆蛋白,LC3-Ⅱ定位于前自噬体和自噬体,LC3-Ⅱ／LC3-Ⅰ比值是自噬体形成数量的重要标志。了解心肌缺血／再灌注时自噬发生的调控机制,有助于认识再灌注损伤的发病机制,并有助于寻找治疗再灌注损伤的有效方法。基于以上理论,本研究初步探讨了心肌缺血／再灌注时自噬的发生时期、自噬在再灌注损伤中的具体作用以及其调控机制。
本实验首先用Langendorff装置制备大鼠离体心脏缺血／再灌注损伤模型,在缺血0、10、20和30 min时和再灌注10、30、60和120 min时采集心脏组织标本,用Western blotting方法检测LC3蛋白的表达。结果显示,在缺血期LC3-Ⅱ／LC3-Ⅰ比值虽有增加趋势,但与对照组相比差异无统计学意义,自噬活动并没有加强。而在再灌注期不同时间点自噬活动均显著加强,说明自噬在心肌再灌注损伤中起一定作用。
为了进一步探讨自噬在再灌注损伤中的确切作用,本研究观察了3-甲基腺嘌呤(3-methyladenine,3-MA)和腺苷受体激动剂(N-Ethylcarboxamidoadenosine, NECA)对自噬和心肌梗死的影响。3-MA为特异性自噬抑制剂,而NECA则为腺苷A2受体拟似剂,是公认的心脏保护物质。再灌注5 min之前开始用3-MA和NECA进行干预并持续35 min。于再灌注10、30、60和120 min时采集心脏组织标本,用Western blotting检测LC3蛋白的表达,以观察3-MA和NECA对自噬的影响。再灌注120 min结束后,TTC染色法测定心肌梗死面积以观察两种干预药物对心肌梗死面积大小的影响,推测自噬在再灌注损伤中的具体作用。结果显示,3-MA抑制再灌注损伤诱发的自噬,并减少心肌梗死面积,说明自噬在心肌再灌注损伤中起有害作用。NECA既能抑制自噬,同时也减轻再灌注引起的心肌梗死。这说明NECA抑制自噬活动可能是其保护再灌注心脏的机制之一。
为了探讨再灌注诱发自噬的机制,本研究用Western blotting方法分别检测了缺血期和再灌注期Beclin 1和mTOR的活性。结果显示,在缺血期和再灌注期,Beclin 1蛋白的表达均没有增加,反而出现了下降的趋势。Beclin 1是自噬正性调节机制中的关键基因,说明在大鼠离体心脏缺血／再灌注损伤模型中,自噬的发生并不主要依赖于其正性调节机制。进一步实验显示,mTOR的活性在缺血期和再灌注期均降低,说明负性调节机制的减弱可能是诱发自噬的原因。
小结
1.在大鼠离体心脏缺血／再灌注损伤模型中,自噬在再灌注期发生；
2.自噬活动在大鼠离体心脏再灌注损伤中起有害作用；
3.在大鼠离体心脏,再灌注诱发的自噬主要是通过负性调节机制引起,其正性调节机制不起主要作用。
Part1
Cyclic guanosine 3'5'-monophosphate (cGMP) is a second messenger molecule and is generated from the catalytic conversion of guanosine triphosphate (GTP) by the soluble guanylyl cyclase (sGC) and the particulate guanylyl cyclase (pGC) As an important cellular messenger, cGMP has three major identified targets: cGMP-dependent protein kinase or protein kinase G (PKG), cGMP-regulated cAMP phosphodiesterase, and cGMP-gated ion channels. Among these targets, PKG has been identified as the most important downstream signal of cGMP. Under physiological conditions, PKG exerts a beneficial influence on the cardiovascular system through relaxation of smooth muscle cells, inhibition of endothelial permeability, inhibition of platelet activation, and negative inotropic effect on cardiomyocytes. Recent studies have shown that the cGMP/PKG signaling pathway is involved in the cardioprotective mechanism underlying ischemic preconditioning and postconditioning. It has also been reported that inhibition of phosphodiesterase-5 with sildenafil (Viagra) which can augment cGMP accumulation protects cardiomyocytes from ischemia/reperfusion injury via a PKG-dependent mechanism. However, the mechanism underlying the cardioprotective effects mediated by the cGMP/PKG signaling pathway remains to be uncertain.
Opening of the mPTP contributes to the pathogenesis of ischemia/reperfusion injury, whereas modulation of the mPTP opening has been proposed to be the common mechanism by which various cardioprotective interventions confer protection against ischemia/reperfusion injury. Although the mPTP has been proposed to be the target of the cGMP/PKG signaling, the impact of the cGMP/PKG on the pore opening is unknown. Studies have shown that glycogen synthase kinase 3β(GSK-3β) may play such a role by interacting with the mPTP after being inactivated by the upstream protective signals. Since sildenafil inactivates GSK-3βvia PKG in cardiomyocytes, it is likely that cGMP may lead to prevention of the mPTP opening by inactivating GSK-3β. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is well known for its negative effect on GSK-3βactivity, and is reported to be activated by sildenafil. Thus, it is possible that the cGMP/PKG signaling inactivates GSK-3βthrough PI3K/Akt. Although Akt activation contributes to cardioprotection of ischemic preconditioning, an excessive activation of Akt also leads to pathologic remodeling and heart failure. Thus, it is intriguing to address the mechanism responsible for the inhibitory effect of cGMP on Akt activity.
The purpose of this study was to determine if cGMP prevents the mPTP opening by inactivating GSK-3(3 in cardiac H9c2 cells and to test if cGMP inactivates GSK-3βby altering Akt activity. To test if cGMP could inactivate GSK-3β, we treated H9c2 cells with the cGMP analogue 8-Br-cGMP (500μmol/L) for 30 min and detected GSK-3P phosphorylation at Ser9 with Western blot. Our data showed that 8-Br-cGMP increased GSK-3βphosphorylation in a dose-dependent manner, indicating cGMP can inactivate GSK-3β. Since PKG is the most important downstream signal of cGMP, we examined the potential role of PKG in the action of 8-Br-cGMP by detecting the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP), a major substrate of PKG. The effect of cGMP on GSK-3βactivity was reversed by the selective PKG inhibitor KT5823 (10μmol/L), indicating that PKG accounts for the action of cGMP. Experiments with cell lysates showed that 8-Br-cGMP alone did not alter but together with PKG la markedly enhanced GSK-3βphosphorylation, confirming that cGMP inhibits GSK-3βthrough PKG. In support, in vitro experiments revealed that interaction of 8-Br-cGMP with PKG la markedly enhanced phosphorylation of purified GSK-3β. This result also indicates that GSK-3βis a direct substrate of PKG. These results clearly demonstrate that cGMP inactivates GSK-3βvia PKG. To determine if cGMP can prevent the mPTP opening, we examined the effect of 8-Br-cGMP on oxidant-induced loss ofΔΨm by monitoring changes in TMRE fluorescence with confocal microscopy. cGMP prevented the loss of TMRE fluorescence caused by H2O2, indicating that cGMP can suppress the mPTP opening. cGMP failed to preserve TMRE fluorescence in cells transfected with the constitutively active GSK-3β(GSK-3P-S9A) mutant plasmid, suggesting that cGMP modulates the mPTP opening by inactivating GSK-3β.
To test if Akt activation contributes to the inhibitory effect of cGMP on GSK-3βactivity, we measured Akt phosphorylation at Ser473 with Western blot. To our great surprise,8-Br-cGMP did not increase but decreased Akt phosphorylation under physiological conditions, indicating that an accumulation of cGMP rapidly inactivates Akt. To confirm this finding, we further tested if the non-specific inhibitor of phosphodiesterases isobutylmethylxanthine (IBMX,200μmol/L) which maintains intracellular cGMP accumulation by inhibiting enzymatic hydrolysis of cGMP, could also mimic the effect of cGMP to inhibit Akt phosphorylation. IB MX markedly reduced Akt phosphorylation but enhanced VASP phosphorylation, indicating that IBMX indeed negatively regulates Akt activity via cGMP. Further experiments showed that 8-Br-cGMP suppresses the effect of insulin on Akt phosphorylation, suggesting that cGMP not only suppresses the basal Akt activity but also impedes the ligand-induced Akt activation. Because an excessive activation of Akt may lead to cardiac hypertrophy and heart failure, our finding suggests that cGMP may prevent hypertrophy and heart failure. The inhibitory effect of cGMP on Akt phosphorylation was not reversed by both the PKG inhibitor KT5823 and PKG siRNA, suggesting that PKG is not required for the negative regulatory action of cGMP on Akt signaling, although it serves as the main downstream signal of cGMP to prevent the mPTP opening by inactivating GSK-3β.
Akt is activated by phosphorylation of Thr308 and Ser473, and dephosphorylation of these residues by Ser/Thr protein phosphatases inactivates the kinase. Thus, we tested if cGMP could inactivate Akt by activating Ser/Thr protein phosphatases. Calyculin A (5 nmol/L), a cell permeable Ser/Thr protein phosphatase inhibitor, dramatically increased Akt phosphorylation, an effect that was partially but significantly blocked by 8-Br-cGMP, indicating that cGMP is able to activate Ser/Thr protein phosphatases, which may lead to inactivation of Akt. To corroborate this finding, we further detected the activity of protein phosphatase 2A (PP2A), a major Ser/Thr protein phosphatase.8-Br-cGMP significantly increased PP2A activity, indicating that cGMP may inhibit Akt activity by activating PP2A.
Summary
1. Cyclic GMP prevents the mPTP opening by inactivating GSK-3βvia PKG in H9c2 cells, which accounts for the cardioprotective of cGMP;
2. Cyclic GMP negatively regulates Akt activity;
3. Cyclic GMP inactivates Akt through activation of PP2A but not through PKG;
4.Cyclkic GMP has duel roles in cardiac survival.
Part1
Ischemic heart disease (IHD) is the leading cause of morbidity and mortality worldwide. The fundamental cause of IHD is myocardial ischemia. Reperfusion therapies cause myocardial contractile dysfunction and tissue damage in the ischemic zone, so-called myocardial reperfusion injury. Development of effective therapies to protect reperfusion injury is an important issue of the medical community. The mechanism underlying myocardial reperfusion injury is complicated and remains unclear, although free radicals, calcium overload, defects in energy metabolism, inflammation, and apoptosis have been proposed to account for the injury. Recent studies indicate that autophagy may play a role in myocardial reperfusion injury. Autophagy occurs constitutively in eukaryotic cells and is a process involving the degradation of cell's own components through the lysosomal machinery. Autophagy occurs under normal conditions, but is also upregulated in response to stress such as starving and hypoxia. Although autophagy can promote cell survival, it may also cause cell death (autophagic cell death or type II programmed cell death). While studies have shown that ischemia/reperfusion can trigger autophagy, the exact induction timing and role of autophagy are unclear. Understanding of the detailed time course for the induction and progression of autophagy during ischemia/reperfusion is critical for the development of preventive and therapeutic interventions for reperfusion injury. Autophagy is regulated by the class I and III of PI3K. The class I inhibits autophagy through mammalian target of rapamycin (mTOR) (the negative pathway), whereas the class III PI3K activates autophagy by increasing Beclin-1 expression, so-called the positive pathway or non mTOR-dependent pathway. LC3, the rat microtubule-associated protein 1 light chain 3, a mammalian homologue of yeast ATG8, is associated with autophagy. There are two forms of LC3, namely, LC3-Ⅰ(cytosol form) and LC3-Ⅱ(processed form). The ratio of LC3-Ⅱ/LC3-Ⅰis correlated with the extent of autophagosome formation and thus was suggested as an excellent marker of autophagy. Investigation of the regulatory mechanism of autophagy occurring during reperfusion is important for understanding of the mechanism underlying reperfusion injury and will help discover effective therapeutic intervention to prevent reperfusion injury. Based on the above theoretical interpretations, the current study investigated the time course of autophagy induction, the exact role of autophagy during reperfusion injury, and the regulatory mechanism underlying the induction of autophagy.
Isolated rat hearts were perfused on a Langendorff apparatus and subjected to 30 min regional ischemia followed by 2 h of reperfusion. Biopsies were collected from the risk zones during ischemia (0,10,20 and 30 min of ischemia) and reperfusion (10,30,60, and 120 min after the onset of reperfusion), and autophagy was determined by the ratio of LC3-Ⅱto LC3-Ⅰwith Western blotting. We found that although LC3-Ⅱ/LC3-Ⅰratio had a tendency to increase during ischemia, there was no statistical difference when compared to the sham group, indicating that autophagy is not prominent during ischemia. In contrast, LC3-Ⅱ/LC3-Ⅰratio was markedly increased upon reperfusion, implying that autophagy may play a role in reperfusion injury.
Since autophagy may play a role during reperfusion, we treated rat hearts with 3-MA and NEC A starting 5 min before the onset of reperfusion for 35 min. Myocardial samples were collected form the risk zones 10,30,60, and 120 min after the onset of reperfusion. Autophagy was determined by the ratio of LC3-Ⅱto LC3-Ⅰwith Western blotting, whereas myocardial infarction was measured with TTC staining. Our data showed that 3-MA not only inhibited autophagy during reperfusion but also reduced infarct size, suggesting that autophagy plays a detrimental role in reperfusion injury. Similarly, NEC A also reduced both autophagy and myocardial infarction, indicating that inhibition of autophagy may contribute to the protective effect of NEC A on reperfusion injury.
To investigate the mechanism underlying the induction of autophagy, we detected Beclin 1 expression and mTOR activities during ischemia and reperfusion with Western blotting. Either ischemia or reperfusion did not enhance Beclin 1 expression, suggesting Beclin 1 may not be critical for the formation of autophagy. Since Beclin 1 is a critical gene for the positive pathway, our observation suggests that the induction of autophagy during reperfusion is not dependent on the positive pathway in isolated rat hearts. In contrast, the activity of mTOR was decreased during both ischemia and reperfusion, indicating that an inhibition of the negative pathway may account for autophagy in the setting of ischemia/reperfusion.
Summary
1. Autophagy is induced upon reperfusion but not during ischemia in isolated rat hearts;
2. Autophagy plays a detrimental role during ischemia/reperfusion;
3. Inhibition of the negative pathway but not the positive pathway account for the formation of autophagy in isolated rat hearts.
引文
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