Pathways of Apoptosis in Hypoxic Cardiac Myocytes

缺氧心肌细胞凋亡途径

• 批准号: 6573154
• 负责人: KEITH A WEBSTER
• 金额: $ 36.74万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6573154
• 关键词: BCL2 gene /protein; JUN kinase; acidity /alkalinity; adenosine triphosphate; apoptosis; calcium flux; cardiac myocytes; cytoprotection; electron transport; enzyme activity; laboratory rat; membrane potentials; mitochondrial membrane; myocardial ischemia /hypoxia; reperfusion

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.

描述(申请人提供)：在所有体外和体内心肌缺血模型中，c-jun氨基末端激酶(JNK)被复氧或再灌注所激活。JNK对细胞存活有很强的调节作用，可能决定再灌流时心肌细胞丢失的程度。关于激活的JNK在不同的再灌注模型中的作用一直存在争议，一些小组描述了促凋亡和其他保护作用。我们提供的新数据表明，JNK功能受到代谢调控，可能解决这一争议；当细胞内[ATP]较高时，JNK具有保护作用，而当[ATP]较低时，JNK具有促凋亡作用。因此，JNK在决定细胞命运的过程中扮演着新陈代谢传感器的角色。我们假设促进生存的目标与促进死亡的目标是不同的。在这项提案的目标1中，我们将使用JNK功能开关作为工具来识别介导这些对低氧心肌细胞的不同影响的磷酸化蛋白。 缺氧和酸中毒是缺血性心脏病的基本特征。这种结合是激活培养中心肌细胞程序性死亡的最强刺激之一。该通路涉及受低氧强烈诱导的BNIP3家族蛋白。含有诱导BNIP3的低氧心肌细胞不会发生程序性死亡，直到环境变得酸性。我们假设BNIP3分别受到缺氧和酸中毒的诱导和激活。建议进行实验以确定酸中毒的机制以及酸中毒是否对线粒体信号有二次影响。由于我们已经确定了BNIP3在培养的心肌细胞中的作用，一个主要的目标是确定在活体内相同的途径是否促进了缺血和脑梗塞期间的细胞死亡。 JNK通过复氧激活的信号尚未确定，但被认为与活性氧物种(ROS)有关。我们的初步数据表明，耦合的线粒体电子传递和膜电位，而不是ROS，是这一信号通路的重要组成部分。我们假设该途径包括由线粒体膜电位激活的钙负荷，以及肌丝钙瞬变。钙被认为激活了PYK2，然后是RAC-1和TAK-1，然后是JNK。用来验证这一假说的实验包括测量缺氧和复氧过程中的膜电位和细胞内钙离子，并将其与Pyk2和JNK联系起来。