Oxidative Stress and Nitric Oxide in Aged Myocardium

这项试点研究资助计划的重点是研究目标2：心血管和脑血管衰老。衰老与许多组织中氧化应激和线粒体功能障碍的增加有关。这些因素被认为在与年龄相关的心室功能、冠状动脉血管储备和对心肌缺血再灌注的耐受性降低中起作用，所有这些都可能导致心血管疾病随着年龄的增加而增加。大量证据表明，线粒体除了维持心室心肌高工作需求的能量储存外，还在决定细胞从可逆损伤到凋亡和坏死的转变中发挥作用。然而，对在体老年心肌缺血-再灌注损伤的研究有限，对老年动物完整心室肌细胞线粒体功能的系统研究较少。该提议将检验老年心肌对缺血-再灌注损伤和氧化应激的耐受性较低的假设，部分原因是一氧化氮（NO）代谢和信号转导的改变。具体目标1将确定来自老年大鼠的体内心肌和分离的心室肌细胞是否表现出对缺血-再灌注的耐受性降低， 缺氧-复氧。具体目标2将检验衰老相关氧化应激和线粒体功能障碍是由于活性氮物质形成增加的假设。在目标1研究中，成年（6个月）和老龄（24个月）Fischer 344 x Brown Norway F1杂种（F344 xBN）大鼠将接受体内冠状动脉闭塞和再灌注，并测量梗死面积。将从成年和老年大鼠分离的左心室肌细胞进行缺氧-再氧合，并测量抽搐幅度和细胞内氧化应激。在目的2研究中，将分离的心室肌细胞暴露于N2 O2以诱导氧化应激。 将在基线条件下以及暴露于H2 O2期间和之后测量线粒体（[Ca 2 +]）、线粒体氧化还原状态和细胞内氧化应激。NO在调节老年心肌细胞对氧化应激的反应中的作用将通过调节NOS活性和精氨酸水平以及NO供体来确定。抗氧化酶和NOS同工型的表达和亚细胞区室化将通过以下方法确定： 亚细胞组分的蛋白质印迹。拟议的研究将提供显着的洞察急性和慢性氧化应激在老年心肌及其调制NO的作用。所获得的结果可能会导致治疗老年人心血管疾病的新疗法的发展。

This pilot research grant proposal focuses on research objective 2: Cardiovascular and Cerebrovascular Aging. Aging is associated with increased oxidative stress and mitochondrial dysfunction in numerous tissues. These factors are thought to play a role in aging-associated decreases in ventricular function, coronary vascular reserve, and tolerance to myocardial ischemia-reperfusion all of which may contribute to the increase in cardiovascular disease with aging. Significant evidence has accumulated that mitochondria, in addition to maintaining the energy stores for the high work demands of ventricular myocardium, play a role in determining the cell's transition from reversible injury to apoptosis and necrosis. However there have been limited studies of ischemia-reperfusion injury in in vivo aged myocardium and few systematic investigations of mitochondrial function in intact ventricular myocytes of aged animals. This proposal will test the hypothesis that aged myocardittm exhibits less tolerance to ischemia-reperfusion injury and oxidative stress in part due to altered nitric oxide (NO) metabolism and signaling. Specific Aim 1 will determine whether in vivo myocardium and isolated ventricular myocytes from aged rats exhibit less tolerance to ischemia-reperfusion and hypoxia-reoxygenation, respectively. Specific Aim 2 will test the hypothesis that aging associated oxidative stress and mitochondrial dysfunction is due to increased formation of reactive nitrogen species. In Aim 1 studies adult (6 month) and aged (24 month) Fischer 344 x Brown Norway F1 hybrid (F344xBN) rats will be submitted to in vivo coronary artery occlusion and reperfusion and infarct size will be measured. Left ventricular myocytes isolated from adult and aged rats will be submitted to hypoxia-reoxygenation and twitch amplitude and intracellular oxidative stress will be measured. In Aim 2 studies isolated ventricular myocytes will be exposed to/-/202 tO induce oxidative stress. Mitochondrial ([Ca2+]), mitochondrial redox state, and intracellular oxidative stress will be measured under baseline conditions and during and following exposure to H202. The role of NO in modulating aged myocyte response to oxidative stress will be determined by modulating NOS activity and arginine levels and with a NO donor. The expression and subcellular compartmentation of anti-oxidant enzymes and NOS isoforms will be determined by Western blotting of subcellular fractions. The proposed studies will provide significant insight into the role of acute and chronic oxidative stress in aged myocardium and its modulation by NO. The results obtained may lead to the development of new therapies for treating cardiovascular disease in the elderly.