小窝蛋白-3在PKC Eplison 介导缺血预适应心脏保护中的作用机制

Caveolin-3 (Cav-3) has been shown to play an important role in the cardioprotection of ischemic preconditioning. Cav-3 which is abundant in caveolae-provides a scaffold to organize, traffic, and regulate signaling molecules. Numerous signaling molecules involved in cardiac protection are known to exist within caveolae or interact directly with caveolin-3. PKC Eplison is one of the most important proteins involved in cardiac protection. Our previous study showed that Cav-3 is colocalized with PKCe in the cell membrane. Activation of adenosine A1 receptors induces translocation of PKCe to the membrane from the cytosol. But whether activated PKCe is binding with Cav-3 is still unknown. We confirmed that Cav-3 binding with endogenous octameric KATP channel in adult rat cardiac myocytes. We hypothesize that the association of KATP channel with Cav-3 plays the important role in PKC-mediated cardioprotection of ischemia preconditioning. The purpose of this study is to elucidate the role of Cav-3 binding with PKCe and to identify signaling mechanisms responsible for PKCe translocation and Cav-3. We will analyze the binding site of Cav-3 with KATP channel by computer software and we investigate that the signal components in cardiac protection coexist and function in the caveolae and that the interaction of signaling molecules with Cav-3 is necessary for cardiac protection.

小窝蛋白-3（Cav-3）在缺血预适应动物模型中显示出心脏保护作用。Cav-3在细胞膜小窝中不仅起支架作用，还能调节信号蛋白的活化，是参与心肌保护作用关键蛋白的交汇点。蛋白激酶Ce(PKCe）在缺血预适应心脏保护中具有重要地位，前期工作证明：Cav-3 和PKCe共表达在细胞膜同一位置，通过调节腺苷A1受体促使PKCe转位进入细胞膜小窝，但PKCe进入小窝后与Cav-3是如何偶联的尚不清楚。已知Cav-3可与PKC信号通路中的KATP通道偶联并调节其活性，推测Cav-3在PKCe介导的缺血预适应中通过与KATP通道偶联发挥心脏保护作用。为了确认这种推测并研究其可能的分子机制，我们拟围绕Cav-3与KATP通道偶联，研究Cav-3与PKCe偶联、活化及转位的关系，利用计算机软件分析Cav-3与KATP通道的偶联活性位点，利用体内外模型揭示心肌缺血预适应中Cav-3与PKCe的偶联机制。

蛋白激酶C（PKC）与小窝蛋白-3（Cav-3）在缺血缺氧预适应动物模型中显示出心脏保护作用，但二者介导的保护作用的确切机制尚不明确，Cav-3在细胞膜小窝中不仅起支架作用，还能调节信号蛋白的活化，是参与心肌保护作用关键蛋白的交汇点。我们通过应用成年大鼠心肌细胞及H9C2细胞系构建细胞缺血缺氧预适应模型，发现预适应可以促使PKCε, PKCδ 和 PKCα从细胞浆内转位至细胞膜Cav-3富集的区域, 而PKCβ和PKCζ不发生以上情况；通过免疫共沉淀实验表明缺氧预适应情况下PKCε, PKCδ or PKCα均可与Cav-3发生偶联；课题组进一步通过荧光共振能量转移实验(fluorescence resonance energy transfer,FRET)发现PKCε-YFP与Cav-3-CFP可以发生FRET现象，表明PKCε在缺氧预适应情况下从细胞浆转位至细胞膜上与Cav-3发生偶联。本课题围绕细胞膜Cav-3与PKC偶联，研究Cav-3与PKCε偶联、活化及转位的关系，进一步利用计算机软件分析Cav-3与PKC的偶联活性位点，利用体内外模型揭示心肌缺血缺氧预适应中Cav-3与PKCε的偶联机制这种心脏保护调节机制。