油酸處理對大鼠心肌細胞間隙接合及肌原纖維之影響

Abstract

心肌缺氧及糖尿病的病人，脂質的代謝平衡受到改變，血液中的不飽和游離脂肪酸濃度會大幅的增加，尤其是油酸(oleic acid)。增高的脂肪酸在心肌細胞內酯化成三酸甘油酯，以脂肪小滴(lipid droplets)的型式堆積在心肌細胞的細胞質。脂肪小滴的堆積會降低心輸出量及干擾間隙接合功能。然而至目前為止，脂肪小滴的堆積對心肌細胞損傷的機制仍不清楚。本研究的第一部分即是探討油酸對培養的新生大白鼠心肌細胞間隙接合的影響及機轉。當細胞以油酸處理後，培養的心肌細胞自發性收縮速率會減低，但不影響細胞存活度。除此之外，位在細胞接合處的Cx43的染色分布也減少，顯示間隙接合的去組合。以scrape loading方法分析間隙接合的功能，結果顯示油酸降低間隙接合所調節的細胞間溝通。已知Cx43的磷酸化可以直接調控間隙接合的組合或去組合。西方墨點分析法(Western blot analysis)証明油酸會引起Cx43 Ser-368磷酸化的增加，這個位置的磷酸化增加已知可能參與間隙接合去組合。Protein kinase C(PKC)參與Cx43 Ser-368磷酸化的增加，因為使用PKC的抑制劑可以阻止油酸引起Cx Ser-368磷酸化的升高。共軛焦的影像顯示PKCe及PKCa分別與Cx43在間隙接合共位(colocalization)，油酸處理後使得位在細胞接合處之PKCe及PKCa分布減少。以含有BSA新鮮培養基置換油酸，繼續培養24小時後，油酸引起兩相鄰細胞間蛋白質分布的改變可回復。進一步以免疫沈澱分析法証實Cx43分別與PKCe及PKCa有生化上的連結，當細胞以油酸處理後，不影響這些蛋白質的交互作用，暗示Cx43與PKCe /PKCa有穩定的連結並導致油酸處理後，因為細胞膜上的間隙接合分布減少，使原本與間隙接合共位的PKCe 及PKCa由細胞膜脫位至細胞質。接著檢驗PKC的異構物(isoforms)，分別加入PKCe以及PKCa的抑制劑，實驗結果發現只有在加入PKCe抑制劑後，可以有效阻止油酸引起的Cx43 Ser-368磷酸化的增加和間隙接合的去組合，並且恢復間隙接合細胞間溝通的能力。以上結果顯示油酸可能藉由活化PKCe，使得Cx43 Ser-368磷酸化的升高，進而導致間隙接合去組合。本研究的第二部分則是觀察油酸對肌原纖維的影響及其機轉。免疫螢螢光染色的結果顯示油酸引發肌節排列紊亂，肌原纖維發生去組合的現象。因為緻密斑(focal adhesions)參與肌原纖維的組合，我們接著檢驗油酸對緻密斑結構的影響。免疫螢光染色的結果顯示，油酸造成integrin b1D、vinculin及paxillin抗體所標示的緻密斑及肋狀體(costameres)結構排列的紊亂及喪失，而且這三個蛋白質的表現均下降。這些變化的時間點早於肌原纖維的去組合，顯示緻密斑參與油酸引發肌原纖維的去組合。將油酸移除後，油酸對於緻密斑及肌原纖維結構的影響可回復。我們進一步檢驗緻密斑蛋白的磷酸化表現。油酸引起FAK與paxillin的酪胺酸去磷酸化，及integrin b1D、paxillin及muscle actin蛋白質表現量下降。而此效應可被酪胺酸去磷酸酶抑制劑原釩酸鈉(sodium orthrovandate)阻斷，同時也可以阻斷油酸引發之肌原纖維去組合，顯示油酸可能活化酪胺酸去磷酸酶。先前的研究指出，FAK及paxillin為PTP-PEST(一種酪胺酸去磷酸酶)的受質，可將此二蛋白去磷酸化。本研究發現油酸引發PTP-PEST蛋白質的向上調節，暗示PPTP-PEST可能參與緻密斑蛋白的去磷酸酸化，引起緻密斑及肋狀體的瓦解，進而導致肌原纖維的去組合。另外，RhoA/cofilin的路徑也可調控肌原纖維的去組合。Cofilin是一種actin-severing蛋白，RhoA會使cofilin的磷酸化增加並抑制其活性，促進actin filament的組合，相反的，RhoA活性降低則減少cofilin磷酸化並引起cofilin活性增加並導致actin filament的去聚合。油酸會減低RhoA的活性及cofilin的磷酸化，此結果暗示油酸導致cofilin活性的增強也許與肌節構造去組合有密切的因果關係。綜合以上的結果，油酸可以透過不同的機制，分別引起間隙接合及肌原纖維的去組合。油酸活化PKCe，引起Cx43 S368磷酸化增加，導致間隙接合去組合。另外，油酸也可能藉由PTP-PEST，參與paxillin及FAK去磷酸化，引起緻密斑去組合，或是直接降低RhoA-cofilin的訊息傳遞，造成actin filaments瓦解，進而引起明帶(I-bands)及其它肌節構造的去組合。

In ischemic and diabetic patients, plasma levels of nonesterified free fatty acids, such as oleic acid (OA), are increased, and the free fatty acids are accumulated as lipid droplets in the cytoplasm of cardiomyocytes, resulting in interference in gap junction-mediated intercellular communication and cardiac output. However, the mechanism of OA-induced electrical uncoupling and contractile dysfunction in cardiomyocytes remains unclear. Therefore, the first section of this study was designed to investigate the effects of OA on gap junctions in cultured neonatal rat cardiomyocytes. OA reduced the spontaneous contraction rates of cultured cardiomyocytes in a time-dependent manner without affecting the cell viability. In addition, Cx43 expression at cell-cell junction decreased after OA treatment, suggesting the disassembly of gap junction. Functional assays by scrape-loading dye transfer assay further demonstrated that OA decreased gap junction-mediated intercellular communication. It is known that Cx43 phosphorylation can modulate the assembly and disassembly of gap junctions. Western blot analysis showed that OA induced Cx43 Ser368 phosphorylation. The phosporylation of this residue has been shown to be involved in gap junction disassembly. PKC participated in Cx43 phosphorylation on Ser368, since PKC inhibitor, calphostin C prevented OA-induced Cx43 Ser368 phosphorylation. Staining for PKCe and PKCa, which were shown to colocalize with Cx43 in confocal images, decreased with increased duration of OA treatment. The effects of OA on these distributional changes at cell junctions were reversed by 24 h incubation in fresh culture medium devoid of OA. Immunoprecipitation assays confirmed the biochemical binding between Cx43 and PKCe/PKCa, and this protein-protein interaction was not affected by OA. This observation may provide the basis for simultaneous detachment of Cx and PKCe/PKCa from the cell-cell junction to the cytosol upon OA stimulation. In order to determine whether PKCε or PKCα was involved in OA-induced Ser368 phosphorylation, we used the PKCε inhibitor, eV1-2, and the PKCα inhibitor, Go6976, and found that only eV1-2 had a significant effect in preventing OA-induced Cx43 Ser368 phosphorylation, gap junction disassembly, and gap junction-mediated intercellular communication. These data suggest that selective activation of PKCε by OA is required for OA-induced Cx43 Ser368 phosphorylation, leading to gap junction disassembly. The second section of this study investigated the effects of OA on contractile apparatus and the signaling pathways involved in this event. OA treatment disrupted myofibrils, as revealed by the disorganization of several sarcomeric proteins. Since focal adhesions (FAs) are implicated in myofibril assembly, we examined structural changes of FAs after OA treatment. Immunofluorescence studies with antibodies against FA proteins (vinculin, integrin b1D, and paxillin) showed that FAs and costameres disintegrated or disappeared after OA treatment and that the changes in FA proteins occurred prior to myofibril disassembly. The effects of OA on FAs and myofibrils were reversed after removal of OA. OA decreased expression of integrin b1D, paxillin, vinculin, and actin, and induced tyrosine dephosphorylation of focal adhesion kinase (FAK) and paxillin. These effects were blocked by pretreatment with sodium orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor. This inhibitor also prevented OA-induced myofibril disassembly, indicating the involvement of PTP in myofibril disassembly. Furthermore, OA increased protein levels of PTP-PEST. The upregulation of this phosphatase correlated with the tyrosine dephosphorylation of paxillin and FAK, which are targets for PTP-PEST. In addition, OA decreased RhoA activity and the phosphorylation of cofilin, a downstream target of RhoA. Cofilin dephosphorylation activated cofilin and led to the depolymerization of F-actin, which might provide the other potential mechanism for myofibril disassembly in addition to FA disassembly. Taken together, OA induced disassembly of gap junctions and myofibrils through different signaling pathways. OA-induced Cx43 Ser368 phosphorylation is mediated by PKCe activation, which might be responsible for OA-induced gap junctional disassembly. In addition, OA also induced tyrosine dephosphorylation of paxillin and FAK by PTP-PEST, which resulted in FA disassembly and subsequent myofibril disruption. In addition, OA induced cofilin activation by decreasing RhoA activity and cofilin phosphorylation levels, which might lead to depolymerization of actin filaments and disassembly of I-bands and other sarcomeric structures.