Bmx Tyrosine Kinase Signaling in Cardiac Protection

描述(由申请人提供)：缺血预适应是防止心脏组织遭受不可逆转的缺血损伤的最有效和持续可重复的方法。这种现象的诱导可以用一氧化氮(NO)供体等药物来替代缺血，从而显著减少心肌梗死范围。已知缺血/再灌注损伤中的心肌细胞死亡与线粒体功能障碍(线粒体通透性转变[MPT])有关，因此需要预防线粒体通透性转变。虽然在心脏保护中防止线粒体功能障碍的信号机制尚不清楚，但基于酪氨酸激酶抑制剂阻断心脏保护的能力，酪氨酸激酶信号转导被认为是可能的。非受体酪氨酸激酶，BMX，在非心肌细胞的细胞保护信号中具有既定的作用，最近在心脏组织中被发现。该蛋白已被证明在非心肌细胞中被PI3-K和Src激活，尽管它在心肌细胞中如何被激活完全未知。我们的初步结果表明，BMX基因敲除(KO)小鼠的心脏不能在没有供体的情况下得到保护，从而暗示BMX是以前未被认识到的保护性信号的必要组成部分。由于这些观察，我们假设BMX信号在心肌细胞中被激活以响应心脏保护并促进细胞存活，至少部分是通过调节线粒体功能。我们提出了两个结合生物化学、蛋白质组分析、共聚焦显微镜和细胞/细胞器生理学来验证这一假说的目标：第一，我们将通过确定PI3-K和Src的作用，识别BMX的激活物，并定位心肌保护信号所需的磷酸化位点，来表征在NO供体诱导的心肌细胞保护过程中BMX的激活。其次，我们将通过测量BMX KO小鼠的线粒体功能，确定该细胞器上的BMX相关蛋白，并确定BMX依赖的线粒体蛋白表达的变化，来评估BMX信号在NO供体诱导的心脏保护中的作用。确定BMX信号机制将首次深入了解心脏中新发现的酪氨酸激酶家族，并有助于更好地了解保护性表型，目标是开发治疗缺血性心脏病的分子方法。

DESCRIPTION (provided by applicant): Ischemic preconditioning is the most potent and consistently reproducible method of preventing heart tissue from undergoing irreversible ischemic damage. Induction of this phenomenon can be recapitulated in lieu of ischemia by drugs such as nitric oxide (NO) donors, which significantly reduce myocardial infarct size. Cardiac cell death in ischemia/reperfusion injury is known to centrally involve malfunction of the mitochondria (mitochondrial permeability transition [MPT]), and protection thus requires prevention of MPT. Although the signaling mechanisms which prevent mitochondrial dysfunction in cardiac protection are unknown, tyrosine kinase signaling has been implicated based on the ability of tyrosine kinase inhibitors to block cardiac protection. The non-receptor tyrosine kinase, Bmx, which has an established role in cytoprotective signaling in non-cardiac cells, was recently identified in cardiac tissue. This protein has been shown to be activated by PI3-K and Src in non-cardiac cells, although how it is activated in the cardiomyocyte is completely unknown. Our preliminary results have shown that the hearts of Bmx knockout (KO) mice cannot be protected with NO donors, thereby implicating Bmx as a previously unrecognized necessary component of protective signaling. Because of these observations, we hypothesize that Bmx signaling is activated in cardiomyocytes in response to cardiac protection and promotes cell survival, at least in part, by regulating mitochondrial function. We propose two Aims that combine biochemistry, proteomic analysis, confocal microscopy and cell/organelle physiology to test this hypothesis: First, we will characterize Bmx activation in mouse cardiomyocytes during NO donor induced protection by determining the role of PI3-K and Src, identifying activators of Bmx and mapping the phosphorylation sites necessary for cardiac protective signaling. Second, we will evaluate the role of Bmx signaling in NO donor induced cardiac protection by measuring mitochondrial function in Bmx KO mice, identifying Bmx associated proteins at this organelle, and determining Bmx-dependent changes in mitochondrial protein expression. Determining Bmx signaling mechanisms will establish the first insights into a newly discovered family of tyrosine kinases in the heart and allow a better understanding of the protective phenotype with the goal of developing molecular approaches to treat ischemic heart disease.