抗氧化蛋白Prx II通过蛋白磷酸化酶1调控PLN磷酸化和心脏泵功能的实验研究

Peroxiredoxin II(PrxII) is one of the members of 2-Cys peroxide peroxidase superfamily in most mammalian cells, which reduces H2O2 or the intracellular peroxides to alcohol and water. Previously, we found that the expression levels of PrxII in the failing human hearts were significantly decreased. In the meantime, the levels were increased in the two functionally hyperdynamic gene engieered mouse hearts, with phospholamban(PLN) knockout or protein phosphostase 1 inhibitor 1 overexpression. However, it remains unclear that the functional significance of PrxII in cardiac contractility and sarcoplasmic reticulum(SR) calcium handling as well as the involvment of PLN. In the current project, we are aiming to evalute the following questions: 1) The role of acute enhanced or reduced expression of PrxII, induced by adenoviral infections, in rat cardiomyocyte contractility and calcium transient and the molecular mechanisms; 2) A transgenic mouse with cardiac specific PrxII overexpression and 3) a Cre-LoxP-induced cardiac specific conditional PrxII deficient mouse will be generated. The cardiac contractile function and calcium transient as well as the SR calcium handing in those two PrxII gene engieered mice will be investigated, under the whole animal, organ and single myocyte levels. 4)The two indicated mice in 2)and 3) will be crossed with PLN knockout mice to generate a double gene altered mouse. The involved cardiac function and SR calcium handling in those animal models will also be determined. 5)In vitro immunoprecipitating study and confocal microscope will be used to further evaluate the interaction between PrxII and several calcium handling proteins, including: PLN, SERCA2 and PP1. 6)Furthermore, the role of the cardiac specific decreased or enhanced expression of PrxII in transaortic banding-induced hypertrophic hearts and the development to heart failure will also be investigated. In summary, the goal of this project is to demonstrate the novel role of PrxII as a regulator for cardiac contractile performance and its pathophysiologic involvement in the progress of heart failure.

Prx II是过氧化氢过氧化物酶超家族成员之一，我们前期研究发现：PrxII在心衰病人的心肌表达量明显下降，而在两个心肌收缩功能增强的小鼠模型：phospholamban(PLN)基因敲除和蛋白磷酸化酶1（PP1）抑制因子1过表达，其表达量上调；但尚不清楚PrxII是否通过PLN调控心肌收缩，本项目旨在探讨：1）腺病毒中介的急性PrxII过表达和表达下调对心肌细胞收缩和肌浆网钙转运的调控；2）慢性PrxII心肌特异性过表达和3）Cre-LoxP中介的条件性PrxII基因敲除对心肌收缩和肌浆网钙转运以及PLN磷酸化的调控；4）将上述PrxII单基因修饰小鼠与PLN基因敲除小鼠交配获得双基因修饰小鼠并检测其心功能；5）确定PrxII与PLN， SERCA2和PP1等收缩蛋白直接相互作用及其6)对病理性心肌肥厚和心衰进程的影响；通过上述研究阐明PrxII可否作为新的心肌收缩调控因子的假说。

研究背景：. 我们前期研究发现：PLN的磷酸化和去磷酸化的动态平衡被蛋白磷酸化酶（protein phosphorylase 1， PP1）和 PP1 抑制蛋白（Inhibitor-1，I-1）调控，进而维持心肌细胞肌浆网的钙稳态和心肌收缩功能的动态平衡，在两个心肌收缩功能均增强的动物模型，即：PLN基因敲除及 I-1活性片段心肌组织过表达的转基因小鼠心脏中，一个存在于心肌细胞胞浆的蛋白质-过氧化氢过氧化物酶 II （Peroxiredoxin II，PrxII）的表达水平均明显上调，但尚不清楚 PrxII 的作用及调控机制。.本项目主要研究内容：.1. 进一步完善腺病毒中介的急性 PrxII 过表达和表达下调对离体大鼠心肌细胞PLN磷酸化，肌浆网钙转运及收缩功能的影响；2. 进一步完善转基因方法中介的慢性心肌特异性 PrxII 过表达对 PLN 磷酸化，肌浆网钙转运和心肌收缩功能的影响；3. 心肌组织 PrxII 过表达小鼠模型的构建及其对 PLN 磷酸化，心肌肌浆网钙转运及心肌收缩功能的影响；4. PrxII 与 PLN，SERCA2，蛋白磷酸化酶1（PP1）以及蛋白磷酸化酶 1 抑制因子 1（I-1）相互作用的实验研究；5. PrxII心肌组织特异性过表达对主动脉缩窄诱导的小鼠实验性心肌肥厚和心力衰竭的影响及其机制研究。.本项目重要结果、关键数据及科学意义.1. 抗氧化蛋白PrxII可与PP1结合。我们利用蛋白免疫共沉淀实验发现：无论是用PrxII 作为诱饵蛋白还是用PP1作为诱饵蛋白，应用心肌匀浆均可发现PrxII可以和PP1在心肌组织相互结合，体外实验结果也如此，因此可知，PrxII对心肌PP1活性的调控作用可能与这两者直接的相互作用相关。.2. PrxII过表达对心肌肥厚至心衰的进程具有保护作用。其机制是通过抑制TAC诱导的PP1活性升高，上调PLN磷酸化蛋白pSer16-PLN和pThr17-PLN表达量进而增强心肌收缩力而实现。.结果首次揭示：在基础状态以及心肌肥厚至心衰进程中，PrxII均可调控心肌收缩功能，对心衰进程具有保护作用，其机制是通过下调 PP1活性，上调PLN磷酸化水平进而增强心肌收缩力实现的。这一发现提示抗氧化蛋白PrxII在心肌肥厚和心力衰竭的治疗中的潜在临床意义。