Pathways of Apoptosis in Hypoxic Cardiac Myocytes

描述（由申请人提供）：C-Jun N-末端激酶（JNK）在所有体外和体内心肌缺血模型中通过复氧或再灌注激活。JNK可以对细胞存活产生强烈的调节作用，并可能决定再灌注期间心肌细胞损失的程度。对于激活的JNK在不同再灌注模型中的作用存在争议，一些组描述了促凋亡和其他保护作用。我们目前的新数据表明，JNK功能的代谢调节，可能会解决这一争议; JNK是保护细胞内[ATP]时，[ATP]是高和促凋亡时，[ATP]低。因此，JNK在决定细胞命运中充当代谢传感器。我们假设促进生存的靶点与促进死亡的靶点不同。在本提案的目标1中，我们将使用JNK功能开关作为工具来鉴定介导这些对缺氧心肌细胞的不同作用的磷酸化蛋白。 缺氧和酸中毒是缺血性心脏病的主要特征。这种组合是激活培养心肌细胞程序性死亡的最强刺激之一。该途径涉及缺氧强烈诱导的Bcl-2家族蛋白BNIP 3。含有诱导的BNIP 3的缺氧心肌细胞不经历程序性死亡，直到环境变得酸性。我们假设BNIP 3分别由缺氧和酸中毒诱导和激活。实验拟确定的机制，以及是否有二次影响线粒体信号酸中毒。由于我们已经确定了BNIP 3在培养的心肌细胞中的作用，因此主要目标是确定在体内缺血和梗死期间是否有相同的途径促进细胞死亡。 JNK通过复氧激活的信号尚未确定，但被认为涉及活性氧（ROS）。我们的初步数据表明，耦合线粒体电子传递和膜电位，但不是ROS是这个信号通路的重要组成部分。我们推测该途径涉及线粒体膜电位激活的钙负荷，再加上肌丝钙瞬变。钙被提议激活激酶Pyk 2，然后是Rac-1和TAK- 1，然后是JNK。实验提出，以测试这一假设，包括测量膜电位和细胞内钙在缺氧和复氧，并将这些Pyk 2和JNK。

DESCRIPTION (provided by applicant): C-Jun N-terminal kinase (JNK) is activated by reoxygenation or reperfusion in all models of myocardial ischemia in vitro and in vivo. JNK can exert strong modulation over cell survival and may determine the extent of myocardial cell loss during reperfusion. Controversy has existed over the role of activated JNK in different models of reperfusion with some groups describing pro-apoptosis and others protection. We present new data demonstrating that JNK functions are metabolically regulated that may resolve this controversy; JNK is protective when intracellular [ATP] is high and pro-apoptotic when [ATP] is low. As such JNK acts as a metabolic sensor in determining cell fate. We hypothesize that the targets that promote survival are different from those that promote death. In aim 1 of this proposal we will use the JNK functional switch as a tool to identify the phospho-proteins that mediate these different effects on hypoxic cardiac myocytes. Hypoxia and acidosis are integral features of ischemic heart disease. This combination is one of the strongest stimuli to activate programmed death of cardiac myocytes in culture. The pathway involves the Bcl-2 family protein BNIP3 that is strongly induced by hypoxia. Hypoxic cardiac myocytes containing induced BNIP3 do not undergo programmed death until the environment becomes acidic. We hypothesize that BNIP3 is induced and activated by hypoxia and acidosis respectively. Experiments are proposed to determine the mechanism and whether there are secondary effects of acidosis on mitochondrial signaling. Because we have established the role of BNIP3 in cultured cardiac myocytes, a major goal is to determine whether the same pathway promotes cell death during ischemia and infarction in vivo. The signals for JNK activation by reoxygenation have not been determined but are thought to involve reactive oxygen species (ROS). Our preliminary data suggest that coupled mitochondrial electron transport and membrane potential, but not ROS are essential components of this signaling pathway. We hypothesize that the pathway involves calcium loading activated by the mitochondrial membrane potential, coupled with myofilament calcium transients. Calcium is proposed to activate the kinase Pyk2 followed by Rac-1 and TAK- 1 and then JNK. Experiments proposed to test this hypothesis include measurements of membrane potential and intracellular calcium during hypoxia and reoxygenation and relating these to Pyk2 and JNK.