整體缺血對大鼠心肌細胞間隙接合之影響:免疫組織化學研究

心肌藉由間隙接合將電流同步性傳播到各心肌細胞，心肌缺血時會出現電流的不通透，近來的研究發現由心肌缺血所引發的電流不通透與間隙接合上組成蛋白質Cx43的磷酸化狀態改變以及Cx43從間隙接合遷移到細胞內部有關。在先前本實驗室以代謝抑制模擬大鼠心肌細胞缺血的研究中顯示，在心肌代謝抑制的過程中lucifer yellow dye coupling降低，同時觀察到Cx43-NP會在間隙接合上聚集並伴隨著Ser368Cx43在間隙接合上的消失。心肌細胞間隙接合的Cx43，會與黏附接合的附屬蛋白質β-catenin、微管的連接蛋白ZO-1以及caveolins之間有直接的連結關係。而目前的研究對於心肌細胞中Cx43及與其交互連結的蛋白質β-catenin、ZO-1及caveolins在心肌缺血時所扮演的角色及相互間如何影響並不清楚。本研究以離體心臟整體缺血灌流的實驗模式使心肌快速缺血，再利用免疫轉漬分析以及免疫螢光染色來觀察整體缺血過程中Cx43、β-catenin、ZO-1及caveolins在心肌細胞中的分布及變化情形。在Cx43的免疫轉漬分析中可觀察到整體缺血的過程伴隨磷酸化Cx43 (44和46 kDa之間)的減少以及Cx43-NP (42kDa) 的增加，接著在雙重免疫螢光的染色中可觀察到Cx43-total整體訊號不變同時伴隨著Cx43-NP的訊號在間隙接合處累積增加，重灌流後Cx43-NP在間隙接合處累積的訊號明顯減少。為了進一步了解整體缺血過程中Cx43的分布變化，我們用雙重免疫螢光染色觀察Ser368Cx43與Cx43-NP在心肌上的分布，結果顯示Ser368Cx43在間隙接合處的訊號減少以及Cx43-NP在間隙接合處的訊號增加，重灌流後則會逆轉上述的現象。接著我們用免疫螢光染色觀察整體缺血過程中與Cx43交互連結的蛋白質β-catenin、ZO-1及caveolins在心肌細胞上的分布情形，其中β-catenin主要分布在細胞接合處，整體缺血過程不影響訊號的強度與分布，而隨著缺血時間的延長p-β-catenin的訊號在細胞質近細胞接合處大量減少，Cx43 -NP在細胞接合處的訊號明顯累積增加。ZO-1主要分布在細胞接合處，與Cx43-total在間隙接合處有同位染色的情形，整體缺血過程不影響ZO-1在間隙接合處訊號的強度與分布。 Caveolin-3主要分布在肌膜與細胞接合處，與Cx43-total在間隙接合處有同位染色的情形。但整體缺血處理之後，隨著缺血時間的增加，細胞接合處上caveolin-3的分布散開且減少，而細胞質裡的點狀染色有增加的情形。至於caveolin-2則分布在心肌細胞以外的區域，整體缺血過程並不影響caveolin-2的染色分布情形。這些結果顯示整體缺血的過程導致Cx43的去磷酸化並伴隨著Cx43-NP在間隙接合處的累積， Ser368Cx43在間盤上的分布減少，p-β-catenin在靠近間盤的細胞質內分布減少，caveolin-3 由細胞表面往細胞內重分布，但對於心肌中ZO-1的染色影響很小。

關鍵字

間隙接合；整體缺血； Cx43

並列摘要

In this study, global ischemia was used to simulate acute myocardial ischemia. Immunoblot analyses and immunofluorescence microscopy were used to determine the distribution of Cx43, β-catenin, ZO-1, and caveolins during global ischemia in cardiomyocytes. Immunoblot analysis of Cx43 showed that ischemia induced a decrease of phosphorylated Cx43 and a concomitant increase of dephosphorylated Cx43(Cx43-NP). Double-labeling immunofluorescence analysis showed that immunoreactivity of Cx43-total has no change and Cx43-NP accumulates in gap junction after ischemia and the accumulation of Cx43-NP was reversed after reperfusion. In order to get a further insight into the distribution of Cx43 during global ischemia in cardiomyocytes, double-labeling immunofluorescence analysis was used to determine the distribution of Ser368Cx43 and Cx43-NP in cardiomyocytes. Global ischemia caused a decrease of Ser368Cx43 decrease and an increase of Cx43-NP in gap junction. These phenomenons were also reversed by reperfusion. Immunofluorescence microscopy was further assessed to observe the distribution of Cx43-associated protein β-catenin, ZO-1, and caveolins in cardiomyocytes.

並列關鍵字

gap junction ； Global Ischemia ； Cx43

參考文獻

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整體缺血對大鼠心肌細胞間隙接合之影響:免疫組織化學研究

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