Yu Hongmei, Yang Zhaogang, Pan Su, Yang Yudan, Tian Jiayi, Wang Luowei, Sun Wei
a Department of Molecular Biology ; College of Basic Medical Sciences; Jilin University ; Changchun; Jilin , China.
b China-Japan Union Hospital; Jilin University ; Changchun; Jilin , China.
Cell Cycle. 2015;14(22):3557-65. doi: 10.1080/15384101.2015.1084446.
Protein kinase C has been shown to play a central role in the cardioprotection of ischemic preconditioning. However, the mechanism underlying PKC-mediated cardioprotection is not completely understood. Given that caveolae are critical for PKC signaling, we sought to determine whether hypoxic preconditioning promotes translocation and association of PKC isoforms with caveolin-3. A cellular model of hypoxic preconditioning from adult rat cardiac myocytes (ARCM) or H9c2 cells was employed to examine PKC isoforms by molecular, biochemical and cellular imaging analysis. Hypoxia was induced by incubating the cells in an airtight chamber in which O2 was replaced by N2 with glucose-free Tyrode's solution. Cells were subjected to hypoxic preconditioning with 10 minutes of hypoxia followed by 30 minutes of reoxygenation. Western blot data indicated that the band intensity for PKCε, PKCδ or PKCα, but not PKCβ and PKCζ was enhanced significantly by hypoxic preconditioning from the caveolin-enriched plasma membrane interactions. Immunoprecipitation experiments from the caveolin-enriched membrane fractions of ARCM showed that the level of PKCε, PKCδ and PKCα in the anti-caveolin-3 immunoprecipitates was also increased by hypoxic preconditioning. Further, our FRET analysis in H9c2 cells suggested that there is a minimum FRET signal for caveolin-3 and PKCε along cell peripherals, but hypoxic preconditioning enhanced the FRET signal, indicating a potential interaction between caveolin-3 and PKCε. And also treatment of the cells with hypoxic preconditioning led to a smaller amount of translocation of PKCε to the mitochondria than that to the membrane. We demonstrate that hypoxic preconditioning promotes rapid association of PKCε, PKCδ and PKCα with the caveolin-enriched plasma membrane microdomain of cardiac myocytes, and PKCε via direct molecular interaction with caveolin-3. This regulatory mechanism may play an important role in cardioprotection.
蛋白激酶C已被证明在缺血预处理的心脏保护中起核心作用。然而，PKC介导的心脏保护的潜在机制尚未完全阐明。鉴于小窝对PKC信号传导至关重要，我们试图确定缺氧预处理是否促进PKC亚型与小窝蛋白-3的转位和结合。采用成年大鼠心肌细胞（ARCM）或H9c2细胞的缺氧预处理细胞模型，通过分子、生化和细胞成像分析来检测PKC亚型。通过将细胞置于气密室中培养诱导缺氧，气密室中用无葡萄糖的台氏液中的N2替代O2。细胞先进行10分钟的缺氧预处理，然后再进行30分钟的复氧。蛋白质印迹数据表明，缺氧预处理可显著增强富含小窝蛋白的质膜相互作用中PKCε、PKCδ或PKCα的条带强度，但PKCβ和PKCζ则不然。来自ARCM富含小窝蛋白的膜组分的免疫沉淀实验表明，缺氧预处理也增加了抗小窝蛋白-3免疫沉淀物中PKCε、PKCδ和PKCα的水平。此外，我们在H9c2细胞中的荧光共振能量转移分析表明，沿着细胞周边，小窝蛋白-3和PKCε存在最小荧光共振能量转移信号，但缺氧预处理增强了荧光共振能量转移信号，表明小窝蛋白-3和PKCε之间存在潜在相互作用。而且，用缺氧预处理处理细胞导致PKCε向线粒体的转位量比向膜的转位量少。我们证明，缺氧预处理促进PKCε、PKCδ和PKCα与心肌细胞富含小窝蛋白的质膜微区快速结合，并且PKCε通过与小窝蛋白-3的直接分子相互作用结合。这种调节机制可能在心脏保护中起重要作用。
