Oxidative Stress and Nitric Oxide in Aged Myocardium

这项试验性研究拨款提案侧重于研究目标2：心血管和脑血管老化。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。这些因素被认为在衰老相关的心功能、冠脉储备和心肌缺血-再灌注耐受性下降中起作用，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，线粒体除了为高工作要求的心肌细胞维持能量储备外，还在决定细胞从可逆性损伤向凋亡和坏死的转变中发挥作用。然而，在体老年心肌缺血再灌注损伤的研究较少，对老年动物完整的心肌细胞线粒体功能的系统研究较少。这一提议将检验一种假说，即老年心肌对缺血再灌注损伤和氧化应激的耐受性较差，部分原因是一氧化氮(NO)代谢和信号的改变。特异性目标1将确定老年大鼠的活体心肌和分离的心室肌细胞是否对缺血-再灌注耐受性较差 分别为缺氧-复氧。特定目标2将检验衰老相关的氧化应激和线粒体功能障碍是由于活性氮物种的增加形成的假说。在目标1研究中，成年(6月龄)和老年(24月龄)Fischer 344×Brown挪威F1杂交(F344xBN)大鼠将被提交给在体冠状动脉阻断和再灌流，并测量梗塞面积。将分离的成年和老年大鼠左室肌细胞置于低氧-复氧状态，并测定其收缩幅度和细胞内氧化应激。在AIM 2研究中，分离的心室肌细胞将暴露于/-/202以诱导氧化应激。 线粒体([Ca~(2+)])、线粒体氧化还原状态和细胞内氧化应激将在基线条件下以及在暴露于H202期间和之后进行测量。NO在调节衰老心肌细胞对氧化应激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。抗氧化酶和一氧化氮合酶亚型的表达和亚细胞划分将由以下因素决定 亚细胞组分的Western blotting。这些研究将对急性和慢性氧化应激在老年心肌中的作用以及NO对其调节作用提供重要的见解。所获得的结果可能会导致开发治疗老年人心血管疾病的新疗法。

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.