抗凋亡蛋白HAX-1心肌损伤保护作用的机制研究

Ischemic cardiac diseases and heart failure are the leading cause of morbidity and mortality in human beings. Cardiomyocyte necrotic and apoptotic loss， as well as sarcoplamic reticulum (SR) pump functional disorder play a key role in mediating the occurrence and development of these lethal cardiac diseases. Recently, we found that the HS-associated protein X-1 (HAX-1), an anti-apoptotic protein, regulates cardiac contractility and SR calcium kinetics through its interactions with sarcoplasmic reticulum calcium-ATPase and phospholambam. In vitro study showed that HAX-1 protected cardiomyocytes from hypoxia-reoxygenation-induced injury. However, the cardioprotection of HAX-1 in vivo, expecially under stress conditions, remains unclear.In the current study, transgenic mice with cardiac specific overexpression of HAX-1 and HAX-1 deficient mice will be used to up-regulate and down-regulate the expression levels of HAX-1 in the heart. Ischemia-reperfusion (I/R)will be performed to induce cardiac injury in vivo. We will evaluate：1）HAX-1 protects hearts from I/R injury in vivo;2)there are specific molecular targets in both mitochondria and endoplasmic reticulum (ER)-dependent cell death pathways in mediating HAX-1's protective effects in the heart, based on the colocalizations of HAX-1 in these two organelles in cardiomyocytes；3)the underlying mechanisms of the interactions between HAX-1 and its molecular targets in both mitochondria and ER against I/R injury; 4)the cardioprotection of HAX-1 depends on its colocalizations of mitochondria and ER. 5)To determine the involvement of SR calcium homeostasis in HAX-1's protection, phospholamban knockout mouse will be mated with HAX-1 overexpressing or deficent mouse to generate a double gene-engineered mouse model.The new model will be used to further evaluate HAX-1's protective effects in the hearts. Our hypothesis is that the cardioprotection of HAX-1 in vivo is associated with the specific interactions between HAX-1 and its molecular targets in both mitochondria and ER. Furthermore, PLN-related calcium homeostasis will play an important role in the crosstalk between these two organells in mediating HAX-1's protective effects. These studies will, for the first time, provide a detail mechanism of HAX-1'cardioprotection in vivo against cardiac injury at subcellular and moleclular levels,indicating HAX-1's beneficial effect to treat ischemic cardiac diseases and heart failure.

HAX-1是与Bcl-2结构具同源性的抗凋亡蛋白,我们前期研究发现：HAX-1同时存在于心肌细胞的线粒体和肌浆网，并可与心肌肌浆网钙泵调控蛋白phospholamban(PLN)相互作用抑制心肌收缩和肌浆网钙转运。本研究利用心肌特异性的HAX-1过表达和HAX-1基因敲除的小鼠模型，以期阐明：1.HAX-1对整体心脏缺血再灌注损伤具有显著保护作用；2.心肌细胞线粒体凋亡和内质网应激通路中均存在与HAX-1保护作用相关的靶分子；3.HAX-1通过与这些靶分子结合并调控其功能发挥心肌保护作用；4.HAX-1的保护作用依赖其在线粒体和肌浆网的双重定位；5.HAX-1与PLN结合对肌浆网钙稳态的调控在其双重心肌保护作用中起重要作用。本研究旨在探讨心肌组织内源性保护因子HAX-1通过线粒体和内质网双重通路发挥保护作用的新机制并论证其双重心肌保护作用假说，为临床缺血性心脏病和心力衰竭提供新的防治策略。

研究背景.我们前期研究发现HAX-1不仅存在于心脏，而且还可以通过与PLN及钙泵的相互作用，调控心肌收缩力以及心肌细胞肌浆网的钙稳态，调节心脏泵功能，HAX-1 表达水平降低与缺血再灌注诱导的心肌细胞损伤相关。.本项目主要研究内容: .1.HAX-1 对整体心脏缺血再灌注损伤具有显著保护作用；2. 心肌细胞线粒体凋亡和内质网应激通路中均存在与HAX-1保护作用相关的靶分子；3.HAX-1 通过与这些靶分子结合并调控其功能发挥心肌保护作用；4.HAX-1 的保护作用依赖其在线粒体和肌浆网的双重定位；5.HAX-1 与 PLN 结合对肌浆网钙稳态的调控在其双重心肌保护作用中起重要作用。探讨HAX-1通过线粒体和内质网双重通路发挥保护作用的新机制并论证其双重心肌保护作用假说，为临床缺血性心脏病和心力衰竭提供新的防治策略。.本项目重要结果、关键数据及科学意义.1）.抗凋亡蛋白HAX-1的表达水平的下降可能参与缺血再灌注所诱导的心肌细胞的损伤。HAX-1对离体及在体心肌缺血再灌注损伤具有明显的保护作用，并可促进心脏收缩功能的恢复，其机制可能与其抗凋亡作用相关，HAX-1通过降低缺血再灌注期的内质网应急以及相关的凋亡通路发挥其保护作用。.2）.cyp-D 的表达下调可能伴随HAX-1的表达水平的上调，HAX-1 过表达可能通过降低 cyp-D 的表达进而降低心肌细胞对钙离子诱导的线粒体膜瞬间通透孔的开放来发挥其对心肌细胞缺血再灌注损伤刺激的保护作用，HAX-1 对心肌线粒体膜瞬间通透孔开放的保护作用的机制可能通过其对 Cyp-D 表达的负调控作用实现，HAX-1 对线粒体 mPTP 开放的调控作用主要依赖于其对 Cyp-D 表达的调控作用，mPTP 的功能在 HAX-1 诱导心肌缺血再灌注损伤的保护作用中起重要作用，HAX-1 过表达可能通过降低线粒体 Cyp-D 与 Hsp90 的相互 作用，促进 Cyp-D 的泛素化降解作用。.我们研究结果首次揭示：HAX-1/Hsp90 复合物可促进 Cyp-D 泛素化降解，从而导致 mPTP 开放受到抑制，进一步阻止氧化应激和线粒体钙超载所致的细胞损伤和死亡。因此， HAX-1 和 Hsp90 可作为一个复合物通过作用于mPTP, 同时抑制细胞的凋亡和坏死，这一发现也同时预测这两个蛋白的相互作用作为临床治疗靶点的潜在治疗意义。

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