摘要:心臟缺血(ischemia)後再灌流/reperfusion 產生的大量的自由基(ROS),可導致心肌細胞之死亡,稱為cardiac I/R injury。在生理狀態下,細胞內free Zn2+i 濃度([Zn2+]i)非常低,大約5 nM。我們初步研究結果發現(Figs.1-6),心肌細胞以I/R 處理,或細胞外加10 μM Zn2+o (in K+ medium),或以Zn2+o 加上pyrithione(Zn2+ ionophore)加入細胞外,皆可引起細胞內[O2-•]i 與[Zn2+]i 大量上升及導致心肌細胞Type-I 細胞凋亡; 細胞內Zn2+-chelator TPEN 可顯著抑制I/R-/Zn2+o-induced [Zn2+]i 大量之上升,並具有強大的心肌細胞保護效果(Figs.1-2)。植物性天然成分的flavonoids:包括baicalein、luteolin、fisetin 等等,亦可顯著抑制由I/R 與Zn2+o 引起之心肌凋亡。如用GSK-3 抑制劑(SB21、LiCl)、p53 抑制劑(PFT-α)或knockdown GSK-3β、p53,對I/R 或Zn2+/K+引起的細胞凋亡,亦有顯著的保護效果。但是在flavonoids、GSK-3β或p53 knockdown 處理下,TPEN仍有顯著的加成保護效果(Figs.2-3);這意謂著有其他ROS-TPEN-/Zn2+i-dependent 路徑存在,而非由GSK-3β-p53 路徑,導致心肌細胞之凋亡(Fig.6D 模式圖)。我們亦發現,在I/R 或Zn2+給予心肌細胞後,可明顯增加proapoptotic NOXAmRN/protein 的表現,anti-apoptotic Mcl-1 protein 減少(Figs. 3D-F)。並且NOXA mRNA 之上升,可被ERK/MEK 抑制劑U0126 所抑制。有趣的是NOXA mRNA 無法被PFT-α所抑制,顯示可能是ERK-NOXA-Mcl-1-dependent,而不是GSK-3ß-p53-dependent 的路徑活化,造成心肌細胞凋亡(Fig. 6D 模式圖)。我們已用大鼠心臟證明,在處理I/R 的同時,給予TPEN 或LiCl,皆可使心肌梗塞之區域明顯下降;令人訝異的是,如將TPEN 與LiCl同給予,仍有加成保護作用(Fig. 4A-B),目前原因尚不明瞭;有待本計劃作進一步研究。我們亦觀察到I/R 處理或外加Zn2+/pyri 處理後,細胞中有大量的vacuoles 增加,LC3-I、LC3-II、p62 及Casp-3 同時增加,如用TPEN chelate Zn2+i 上升;或用antioxidantNAC、DTT 抑制細胞中ROS 產生,則可明顯抑制LC3-I、LC3-II、p62 及Casp-3 的上升及增加心肌細胞存活(Figs.2-3 ; 5-6);是否autophagy(自我吞噬)與apoptosis(凋亡)有上下游之關係?值得進一步研究。奇怪的是,p62 明顯上升,一般認為是抑制autophagy,而LC3-II (aggregate form)上升,乃是促進autophagy;為何I/R 處理導致LC3-II 及p62同時上升的矛盾現象出現?目前須進一步探討。Zn2+chelator (TPEN)與antioxidants(MnTBAP、NAC、DTT)具有不同藥理作用;有趣的是,它們皆具類似效果:因它們(i).皆可抑制I/R-induced LC3-I、LC3-II 及p62 等蛋白質的表現(Figs.5, 6A-B) ;(ii).亦可明顯抑制I/R-induced LC3A/B-GFP 過度表現(Fig. 6C);(iii). 且皆可增加細胞存活(Fig.2)。上述特異有趣的現象有待本計劃作進一步釐清。第一年及第二年計畫: 以初級培養之幼鼠心肌細胞及cardiomyoblasts,進行autophagy之研究。(A). 我們將繼續深入探討I/R 所引起的autophagy 是否與apoptosis 有上下游之關係?(B). 探討autophagy 與apoptosis 之間可能的交互作用(cross-talk)及相關蛋白質(cross-talk protein) ;並研究其分子機制。(C). 探討GSK-3β-p53 及/或NOXA-Mcl-1-dependent signaling 是否是引起autophagy的原因之一?(D). 探討I/R 是否可引起心肌細胞粒腺體中ROS 上升,而造成mitophagy?(E). 探討I/R 可否引起心肌細胞ER-stress,而造成ER-phagy 之可能性。(F). 探討在I/R 或Zn2+/pyri 處理後,同時有LC3-I、LC3-II 及p62 上升,此有趣且相互矛盾的現象之分子機制為何?(G).探討p62 堆積現象,在I/R injury 可能扮演的嶄新角色。第三年計劃:(A). 以in vivo 動物實驗模式,測試小鼠心臟I/R 處理後,藥物對心臟的保護作用及其相關的分子機制。由於處理TPEN 或三種植物性天然成分的flavonoids,已明顯改善幼鼠心肌細胞,cardiomyoblasts 或大鼠離體心臟的I/R injury(Figs.1-4),至今尚未有任何論文報告過。加上本計劃第一年及第二年在autophagy 之研究發現,可能參與I/R injury 嶄新的autophagy 蛋白質,值得進一步以in vivo 動物模式測試;觀察以低劑量給予各種心臟保護藥物,在in vivo 情況下之心臟保護效果;所得的結果,可能將對I/R 造成之心肌損傷有相當重要性及貢獻。(B).以初級培養之心肌細胞,進行DNA microarray 研究。找出除了GSK-3ß、p53、NOXA 及autophagy proteins 等蛋白質參與心肌死亡的分子機制; 將深入探討有無其它 novel 及autophagy/apoptosis proteins 之參與,以作為對抗心肌缺血-再灌流而造成之心肌死亡之參考。
Abstract: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are both suggested to beinvolved in cardiac ischemia-reperfusion (I/R) injury. However, it remains unclear themolecular mechanism for the I/R-induced intracellular Zn2+ (Zn2+i) release resulting incardiac I/R injury. Our preliminary results (Figs. 1-6) showed that, when myocytes orcardiomyoblasts were exposed to I/R, 10 μM Zn2+o/K+ or 10 μM Zn2+o/pyri, a markedincrease in the [O2-•]i and [Zn2+]i were both seen (Fig. 1). TPEN (a high affinityintracellular Zn2+ chelator) markedly abolished the I/R- or Zn2+o-induced Zn2+i release andprotected against I/R- or Zn2+o-induced caspase-3-dependent myocyte apoptosis(Figs.1-2). Interestingly, U0126 (MEK/ERK inhibitor) and three potent flavonoids (baicalein,luteolin and fisetin), had little effect against the increase in the [O2-•]i and [Zn2+]i (Fig. 1B),while they all markedly prevented the I/R- and Zn2+o-induced caspase-3-dependentapoptosis (Fig. 2), indicating that activation of the flavonoids-sensitive target proteinoccurs downstream of the O2-•-/Zn2+i-increase-induced apoptosis. LiCl, SB21 (GSK-3inhibitors) and PFT-α (p53 inhibitor; p53 is one of GSK-3 substrates) had only partialprotective effect (Figs. 2-3). Similar results for partial cardioprotection were also seenwhen knockdown either GSK-3β or p53 protein, suggesting that activation of theGSK-3β-p53 axis was involved in the I/R injury. We also showed that I/R or Zn2+/pyriinduced an increase in levels of NOXA mRNA and pro-apoptotic NOXA protein and adecrease in anti-apoptotic Mcl-1 protein, which were both not sensitive to GSK-3β or p53inhibition (Fig. 3). Furthermore, knockdown of NOXA also had partial protective effectagainst the I/R- and Zn2+o-induced myocyte apoptosis (Figs. 3; 6D). We thus suggested thatERK-NOXA-Mcl-1 and ERK-GSK-3β-p53-dependent signaling axies were both involedin the cardiac I/R injury (Fig. 6D). Interestingly, there was a significant additionalcardioprotection by TPEN in the presence of SB21, LiCl, siGSK-3β, sip53, PTF-α orflavonoids (Figs. 2-4); i.e. TPEN was always the most potent protective drug than abovetreatments in neonatal cardiolmyocytes, cardiomyoblasts and adult rat isolated hearts. We willfurther investigate this interesting point in the future study.After exposure to the I/R or Zn2+/pyri, however, we also observed that there were largeamount of vesicles appeared in cytosol, together with a time-dependent increase in animportant autophagy marker, aggregate form of LC3-II (Figs. 5A;6A). However, it ispuzzlingly that there was simultaneous increase in the levels of LC3-I and p62. Interestingly,when LC3-I, LC3-II and p62 increased at 3-6 hr after the I/R or Zn2+/pyri teatment, therewere followed by increase in levels of caspase-3 and PARP-1 (apoptosis markers).Interestingly, both the autophagy and apoptosis makers remain elevated at 72 hr after thetreatment (Figs. 5A; 5E). In addition, after over-expression of LC3A/B-GFP, the I/Rexposure again significantly induced a simultaneous increase in the activation of bothLC3-II and Casp-3 (Fig. 6C). Intriguingly, LC3-I, LC3-II and p62 was all abolished byTPEN, NAC or DTT, but not by BA1, 3-MA or z-VAD (Figs. 5-6).We will perform more detail studies regarding the following subjects:(I). In vitro study (primary cultured neonatal cardiomyocytes and cardiomyoblasts):(A). To investigate whether the TPEN-/Zn2+i -sensitive autophagy may be involved in the I/Rinjury?(B). (i). Whether the autophagy may be an upstream signaling for the I/R-induced apoptoicpathway? (ii). To investigate possible interactions between the autophagy pathway and theapoptosis pathway. (iii). To investigate the cross-talk proteins.(C). To investigate molecular mechanism why there were simultaneously increases in levelsof TPEN-/antioxidants-sensitive LC3-I , LC3-II and p62?(II). In vivo study (in vivo mice coronary LAD ligation; induction of cardiac I/R injury):(A). To test all the protective chemicals in the mice heart for a longer lifespan of the mice invivo, since there are shorter lifespan (3-4 days) of the primary cardiomyocytes (Figs. 1-3) andof the rat isolated heart (~2 hr, Fig. 4). The tested drugs are: low dosage of TPEN, flavonoids,U0126, and autophagy proteins/inhibitors.(B). (i). To find out which drug is the most potent in the cardioprotection. (ii). To investigatepossible target protein for the protective drug.(III). In vitro study (primary cultured neonatal cardiomyocytes): If all the target proteinshave only partial protective effect against oxidative stress-induced myocyte death, DNAmicroarray will be performed to find out novel proteins, which may be involved in the cardiacI/R injury or in the cardioprotection.